Tissue-homing peptide conjugates and methods of use thereof

ABSTRACT

Peptides that home, target, migrate to, are directed to, are retained by, or accumulate in and/or bind to the cartilage or kidney of a subject are disclosed. Pharmaceutical compositions and uses for peptides or peptide-active agent complexes comprising such peptides are also disclosed. Such compositions can be formulated for targeted delivery of an active agent to a target region, tissue, structure or cell in the cartilage. Targeted compositions of the disclosure can deliver peptide or peptide-active agent complexes to target regions, tissues, structures, or cells targeted by the peptide.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/409,661, filed Aug. 23, 2021, which is a continuation-in-part of U.S.patent application Ser. No. 17/230,353, filed April 14, 2021, which is acontinuation of U.S. patent application Ser. No. 16/492,914, filed Sep.10, 2019, now U.S. Pat. No. 11,013,814, which is a national stage entryof PCT Application No. PCT/US2018/023006, filed Mar. 16, 2018, whichclaims the benefit of U.S. Provisional Patent Application No.62/472,485, filed Mar. 16, 2017, and U.S. patent application Ser. No.17/409,661 is a continuation-in-part of U.S. patent application Ser. No.16/704,955, filed Dec. 5, 2019, which is a divisional of U.S. patentapplication Ser. No. 14/855,355, filed Sep. 15, 2015, now abandoned,which is a continuation of International Application No.PCT/US2014/056177, filed Sep. 17, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/990,101, filed May 7, 2014, U.S.Provisional Patent Application No. 61/879,108, filed Sep. 17, 2013, andU.S. Provisional Patent Application No. 61/879,096, filed Sep. 17, 2013,which are incorporated herein by reference in their entirety for allpurposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentby the National Cancer Institute, National Institutes of Health,Department of Health and Human Services, under Contract No.HHSN261201200054C.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on July 22, 2021, isnamed “109561-691093_SL.txt” and is 547,229 bytes in size.

BACKGROUND OF THE INVENTION

Cartilage comprises chondrocytes, a specialized cell-type which producescomponents of the extracellular matrix, mainly including collagen,proteoglycans (e.g., aggrecan), and elastic fibers. The extracellularmatrix proteins provide support, cushion, and durability tocartilage-rich portions of the body such as joints, ears, nose, andwindpipe. Cartilage is one of few tissues in the body which does notcontain blood vessels and is considered an avascular tissue. Unlike manycells in the body which rely on a combination of blood flow anddiffusion, chondrocytes rely on diffusion. Because it does not have adirect blood supply, compared to other connective tissues, cartilagegrows and repairs much more slowly. As a result, cartilage disorders areparticularly difficult to treat.

Furthermore, for many types of cancer, the precision of surgicalresection directly influences patient prognosis. Unfortunately,intra-operative identification of tumor margins or small foci of cancercells remains imprecise or depends on surgical judgment. Thus, theextent of surgical resection is constrained by the requirement to avoidharming vital structures.

Despite the advances in the development of probes for targeting andimaging tumors, there exists a need for a probe that allows forintra-operative visualization of cancerous tissues.

SUMMARY OF THE INVENTION

The present disclosure relates to compositions and methods for treatmentof cartilage disorders. Described herein are peptides that home to,migrate to, accumulate in, bind to, are retained by, or are directed to,and/or bind in cartilage following administration in a subject. In someembodiments, the homing peptides of the present disclosure are used todeliver a detection agent to image and/or diagnose cartilage, injury, ordisease. In some embodiments, compositions and methods for treatment ofkidney disorders are described. In other embodiments, the homingpeptides of the present disclosure are used to treat or deliver anactive agent to a region, tissue, structure, or cell thereof

In some aspects, a peptide active agent conjugate comprises: a) apeptide, wherein the peptide comprises a sequence that has at least 70%sequence identity with any one of SEQ ID NO: 508—SEQ ID NO: 758 and uponadministration to a subject the peptide homes, targets, migrates to,accumulates in, binds to, is retained by, or is directed to a cartilageof the subject, and an active agent selected from an active agent classselected from TABLE 53 or TABLE 55; b) a peptide, wherein the peptidecomprises a sequence that has at least 70% sequence identity with anyone of SEQ ID NO: 508—SEQ ID NO: 758 and upon administration to asubject the peptide homes, targets, migrates to, accumulates in, bindsto, is retained by, or is directed to a kidney of the subject, and anactive agent selected from an active agent class selected from TABLE 54or TABLE 55; c) a peptide, wherein the peptide comprises a sequence thathas at least 70% sequence identity with any one of SEQ ID NO: 508—SEQ IDNO: 758 and upon administration to a subject the peptide homes, targets,migrates to, accumulates in, binds to, is retained by, or is directed toa cartilage or kidney of the subject, and an active agent selected fromTABLE 53, TABLE 54, or TABLE 55; d) a peptide, wherein the peptidecomprises a sequence that has at least 70% sequence identity with anyone of SEQ ID NO: 798—SEQ ID NO: 1048 and upon administration to asubject the peptide homes, targets, migrates to, accumulates in, bindsto, is retained by, or is directed to a cartilage of the subject, and anactive agent selected from TABLE 53 or TABLE 55; e) a peptide, whereinthe peptide comprises a sequence that has at least 70% sequence identitywith any one of SEQ ID NO: 798—SEQ ID NO: 1048 and upon administrationto a subject the peptide homes, targets, migrates to, accumulates in,binds to, is retained by, or is directed to a kidney of the subject, andan active agent selected from an active agent class selected from TABLE54 or TABLE 55; f) a peptide, wherein the peptide comprises a sequencethat has at least 70% sequence identity with any one of SEQ ID NO:798—SEQ ID NO: 1048 and upon administration to a subject the peptidehomes, targets, migrates to, accumulates in, binds to, is retained by,or is directed to a cartilage or kidney of the subject, and an activeagent selected from an active agent class selected from TABLE 53, TABLE54, or TABLE 55; g) a peptide, wherein the peptide comprises a sequencethat has at least 70% sequence identity with any one of SEQ ID NO:744—SEQ ID NO: 758 and upon administration to a subject the peptidehomes, targets, migrates to, accumulates in, binds to, is retained by,or is directed to a cartilage of the subject, and an active agentselected from TABLE 53, TABLE 55, or TABLE 56; h) a peptide, wherein thepeptide comprises a sequence that has at least 70% sequence identitywith any one of SEQ ID NO: 744—SEQ ID NO: 758 and upon administration toa subject the peptide homes, targets, migrates to, accumulates in, bindsto, is retained by, or is directed to a kidney of the subject, and anactive agent selected from TABLE 54, TABLE 55, or TABLE 56; i) apeptide, wherein the peptide comprises a sequence that has at least 70%sequence identity with any one of SEQ ID NO: 744—SEQ ID NO: 758 and uponadministration to a subject the peptide homes, targets, migrates to,accumulates in, binds to, is retained by, or is directed to a cartilageor a kidney of the subject, and an active agent selected from TABLE 53,TABLE 54, TABLE 55, or TABLE 56; j) a peptide, wherein the peptidecomprises a sequence that has at least 70% sequence identity with anyone of SEQ ID NO: 1034 — SEQ ID NO: 1048 and upon administration to asubject the peptide homes, targets, migrates to, accumulates in, bindsto, is retained by, or is directed to a cartilage of the subject, and anactive agent selected from TABLE 53, TABLE 55, or TABLE 56; k) apeptide, wherein the peptide comprises a sequence that has at least 70%sequence identity with any one of SEQ ID NO: 1034—SEQ ID NO: 1048 andupon administration to a subject the peptide homes, targets, migratesto, accumulates in, binds to, is retained by, or is directed to a kidneyof the subject, and an active agent selected from TABLE 54, TABLE 55, orTABLE 56; or 1) a peptide, wherein the peptide comprises a sequence thathas at least 70% sequence identity with any one of SEQ ID NO: 1034—SEQID NO: 1048 and upon administration to a subject the peptide homes,targets, migrates to, accumulates in, binds to, is retained by, or isdirected to a cartilage or a kidney of the subject, and an active agentselected from TABLE 53, TABLE 54, TABLE 55, or TABLE 56. In someembodiments, the peptide comprises: a) a sequence that has at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 99% or 100% sequence identity with any one of SEQ ID NO: 508—SEQID NO: 758 or a fragment thereof; b) a sequence that has at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 99% or 100% sequence identity with any one of SEQ ID NO: 744—SEQID NO: 758 or a fragment thereof; c) a sequence that has at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 99% or 100% sequence identity with any one of SEQ ID NO: 798—SEQID NO: 1048 or a fragment thereof; or d) a sequence that has at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99% or 100% sequence identity with any one of SEQ ID NO:1034—SEQ ID NO: 1048 or a fragment thereof. In some embodiments, thepeptide comprises: a) a sequence of any one of SEQ ID NO: 508—SEQ ID NO:758 or a fragment thereof; b) a sequence of any one of SEQ ID NO:744—SEQ ID NO: 758 or a fragment thereof; c) a sequence of any one ofSEQ ID NO: 798—SEQ ID NO: 1048 or a fragment thereof; or d) a sequenceof any one of SEQ ID NO: 1034—SEQ ID NO: 1048 or a fragment thereof.

In some aspects, a peptide comprises a sequence that has at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99%, or 100% sequence identity with any one of SEQID NO: 744—SEQ ID NO: 758 or at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or100% sequence identity with any one of SEQ ID NO: 1034—SEQ ID NO: 1048.

In some embodiments, the peptide comprises: a) a sequence of any one ofSEQ ID NO: 485—SEQ ID NO: 507 or a fragment thereof; b) a sequence ofany one of SEQ ID NO: 759—SEQ ID NO: 781 or a fragment thereof; c) asequence of any one of SEQ ID NO: 50—SEQ ID NO: 507 or a fragmentthereof; or d) a sequence of any one of SEQ ID NO: 779—SEQ ID NO: 781 ora fragment thereof. In some embodiments, the peptide is at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least, 97%, at least 98%, or at least 99%identical to any one of SEQ ID NO: 978—SEQ ID NO: 1024 or at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 97%, at least 98%, or at least 99%identical to any one of SEQ ID NO: 688—SEQ ID NO: 734. In someembodiments, the peptide is at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, or 100% identical to: a) SEQ ID NO: 595; b) SEQID NO: 885; c)SEQ ID NO: 508 SEQ ID NO: 508; d) SEQ ID NO: 798; e) SEQID NO: 511; SEQ ID NO: 801; g) SEQ ID NO: 669; h) SEQ ID NO: 959; i) SEQID NO: 514; j) SEQ ID NO: 804; k) SEQ ID NO: 592; 1) SEQ ID NO: 882; m)SEQ ID NO: 520; n) SEQ ID NO: 810; o) SEQ ID NO: 683; p) SEQ ID NO: 962;q) SEQ ID NO: 509; r) SEQ ID NO: 799; s) SEQ ID NO: 590; t) SEQ ID NO:880; u) SEQ ID NO: 510; v) SEQ ID NO: 800; w) SEQ ID NO: 671; x) SEQ IDNO: 961; y) SEQ ID NO: 591; or z) SEQ ID NO: 881. In some embodiments,the peptide is at least 70%, at least 80%, at least 90%, at least 95%,at least 97%, at least 99%, or 100% identical to: a) SEQ ID NO: 1034; b)SEQ ID NO: 1035; c) SEQ ID NO: 1036; d) SEQ ID NO: 1037; e) SEQ ID NO:1038; f) SEQ ID NO: 1039; g) SEQ ID NO: 1040; h) SEQ ID NO: 1041; i) SEQID NO: 1042; j) SEQ ID NO: 1043; k) SEQ ID NO: 1044; 1) SEQ ID NO: 1045;m) SEQ ID NO: 1046; n) SEQ ID NO: 1047; o) SEQ ID NO: 1048; p) SEQ IDNO: 744; q) SEQ ID NO: 745; r) SEQ ID NO: 746; s) SEQ ID NO: 747; t) SEQID NO: 748; u) SEQ ID NO: 749; v) SEQ ID NO: 750; w) SEQ ID NO: 751; x)SEQ ID NO: 752; y) SEQ ID NO: 753; z) SEQ ID NO: 754; aa) SEQ ID NO:755; bb) SEQ ID NO: 756; cc) SEQ ID NO: 757; or dd) SEQ ID NO: 758. Insome embodiments, the peptide homes, targets, migrates to, accumulatesin, binds to, is retained by, or is directed to cartilage, to kidney, orto cartilage and kidney. In some embodiments, the peptide homes,targets, migrates to, accumulates in, binds to, is retained by, or isdirected to proximal tubules of the kidney. In some embodiments, thepeptide is covalently conjugated to the active agent. In someembodiments, the peptide active agent conjugate homes, targets, migratesto, accumulates in, binds to, is retained by, or is directed to acartilage or a kidney of the subject.

In some embodiments, the peptide comprises 4 or more cysteine residues.In some embodiments, the peptide comprises three or more disulfidebridges formed between cysteine residues, wherein one of the disulfidebridges passes through a loop formed by two other disulfide bridges. Insome embodiments, the peptide comprises a plurality of disulfide bridgesformed between cysteine residues. In some embodiments, the peptidecomprises a disulfide through a disulfide knot. In some embodiments, atleast one amino acid residue of the peptide is in an L configuration or,wherein at least one amino acid residue of the peptide is in a Dconfiguration.

In some embodiments, the sequence comprises at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 26, at least 27, at least 28, at least29, at least 30, at least 31, at least 32, at least 33, at least 34, atleast 35, at least 36, at least 37, at least 38, at least 39, at least40, at least 41, at least 42, at least 43, at least 44, at least 45, atleast 46, at least 47, at least 48, at least 49, at least 50, at least51, at least 52, at least 53, at least 54, at least 55, at least 56, atleast 57, at least 58 residues, at least 59, at least 60, at least 61,at least 62, at least 63, at least 64, at least 65, at least 66, atleast 67, at least 68, at least 69, at least 70, at least 71, at least72, at least 73, at least 74, at least 75, at least 76, at least 77, atleast 78, at least 79, at least 80, or at least 81 residues.

In some embodiments, any one or more K residues are replaced by an Rresidue or wherein any one or more R residues are replaced by a Kresidue. In some embodiments, any one or more M residues are replaced byany one of the I, L, or V residues. In some embodiments, any one or moreL residues are replaced by any one of the V, I, or M residues. In someembodiments, any one or more I residues are replaced by any of the M, L,or V residues. In some embodiments, any one or more V residues arereplaced by any of the M, I, or L residues. In some embodiments, any oneor more G residues are replaced by an A residue or wherein any one ormore A residues are replaced by a G residue. In some embodiments, anyone or more S residues are replaced by a T residue or wherein any one ormore T residues are replaced by for an S residue. In some embodiments,any one or more Q residues are replaced by an N residue or wherein anyone or more N residues are replaced by a Q residue. In some embodiments,any one or more D residues are replaced by an E residue or wherein anyone or more E residues are replaced by a D residue.

In some embodiments, the peptide has a charge distribution comprising anacidic region and a basic region. In some embodiments, the acidic regionis a nub. In some embodiments, the basic region is a patch. In someembodiments, the peptide comprises 5-12 basic residues. In someembodiments, the peptide comprises 0-5 acidic residues. In someembodiments, the peptide comprises 6 or more basic residues and 2 orfewer acidic residues. In some embodiments, the peptide comprises a 4-19amino acid residue fragment containing at least 2 cysteine residues, andat least 2 positively charged amino acid residues. In some embodiments,the peptide comprises a 20-70 amino acid residue fragment containing atleast 2 cysteine residues, no more than 2 basic residues and at least 2positively charged amino acid residues. In some embodiments, the peptidecomprises at least 3 positively charged amino acid residues. In someembodiments, the positively charged amino acid residues are selectedfrom K, R, or a combination thereof.

In some embodiments, the peptide has a charge greater than 2 atphysiological pH. In some embodiments, the peptide has a charge greaterthan 3.5 at physiological pH. In some embodiments, the peptide has acharge greater than 4.5 at physiological pH. In some embodiments, thepeptide has a charge greater than 5.5 at physiological pH. In someembodiments, the peptide has a charge greater than 6.5 at physiologicalpH. In some embodiments, the peptide has a charge greater than 7.5 atphysiological pH. In some embodiments, the peptide has a charge greaterthan 8.5 at physiological pH. In some embodiments, the peptide has acharge greater than 9.5 at physiological pH.

In some embodiments, the peptide is selected from a potassium channelagonist, a potassium channel antagonist, a portion of a potassiumchannel, a sodium channel agonist, a sodium channel antagonist, acalcium channel agonist, a calcium channel antagonist, a hadrucalcin, atheraphotoxin, a huwentoxin, a kaliotoxin, a cobatoxin, or a lectin. Insome embodiments, the lectin is SHL-Ib2.

In some embodiments, the peptide is arranged in a multimeric structurewith at least one other peptide.

In some embodiments, at least one residue of the peptide comprises achemical modification. In some embodiments, the chemical modification isblocking the N-terminus of the peptide. In some embodiments, wherein thechemical modification is methylation, acetylation, or acylation. In someembodiments, the chemical modification is: methylation of one or morelysine residues or analogue thereof; methylation of the N-terminus; ormethylation of one or more lysine residue or analogue thereof andmethylation of the N-terminus. In some embodiments, the peptide islinked to an acyl adduct.

In some embodiments, the peptide is linked to an active agent. In someembodiments, the active agent is fused with the peptide at an N-terminusor a C-terminus of the peptide. In some embodiments, the active agent isanother peptide. In some embodiments, the active agent is an antibody.In some embodiments, the active agent is an Fc domain, Fab domain, scFv,or Fv fragment. In some embodiments, the peptide fused with an Fc domaincomprises a contiguous sequence. In some embodiments, 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 active agents are linked to the peptide. In someembodiments, the peptide is linked to the active agent at an N-terminus,at the epsilon amine of an internal lysine residue, at the carboxylicacid of an aspartic acid or glutamic acid residue, or a C-terminus ofthe peptide by a linker. In some embodiments, the peptide is linked tothe active agent via a cleavable linker. In some embodiments, thepeptide or peptide active agent conjugate further comprises anon-natural amino acid, wherein the non-natural amino acid is aninsertion, appendage, or substitution for another amino acid.

In some embodiments, the peptide is linked to the active agent at thenon-natural amino acid by a linker. In some embodiments, the linkercomprises an amide bond, an ester bond, a carbamate bond, a carbonatebond, a hydrazone bond, an oxime bond, a disulfide bond, a thioesterbond, a thioether bond, a triazole, a carbon-carbon bond, or acarbon-nitrogen bond. In some embodiments, the cleavable linkercomprises a cleavage site for matrix metalloproteinases, thrombin,cathepsins, or beta-glucuronidase. In some embodiments, the linker is ahydrolytically labile linker. In some embodiments, the linker is pHsensitive, reducible, glutathione-sensitive, or protease cleavable. Insome embodiments, the peptide is linked to the active agent via a stablelinker. In some embodiments, the peptide has an isoelectric point ofabout 9.

In some embodiments, the peptide is linked to a detectable agent. Insome embodiments, the detectable agent is fused with the peptide at anN-terminus or a C-terminus of the peptide. In some embodiments, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 detectable agents are linked to the peptide. Insome embodiments, the peptide is linked to the detectable agent via acleavable linker. In some embodiments, the peptide is linked to thedetectable agent at an N-terminus, at the epsilon amine of an internallysine residue, or a C-terminus of the peptide by a linker. In someembodiments, the peptide active agent conjugate or peptide furthercomprises a non-natural amino acid, wherein the non-natural amino acidis an insertion, appendage, or substitution for another amino acid.

In some embodiments, the peptide is linked to the detectable agent atthe non-natural amino acid by a linker In some embodiments, the linkercomprises an amide bond, an ester bond, a carbamate bond, a hydrazonebond, an oxime bond, or a carbon-nitrogen bond. In some embodiments, thecleavable linker comprises a cleavage site for matrixmetalloproteinases, thrombin, cathepsins, or beta-glucuronidase. In someembodiments, the peptide is linked to the detectable agent via a stablelinker. In some embodiments, the detectable agent is a fluorophore, anear-infrared dye, a contrast agent, a nanoparticle, a metal-containingnanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, aradioisotope, or a radionuclide chelator. In some embodiments, thedetectable agent is a fluorescent dye.

In some aspects, a pharmaceutical composition comprises the peptideactive agent conjugate of any embodiment as described herein or a saltthereof, or the peptide of any embodiment as described herein or a saltthereof, and a pharmaceutically acceptable carrier. In some embodiments,the pharmaceutical composition is formulated for administration to asubject. In some embodiments, the pharmaceutical composition isformulated for inhalation, intranasal administration, oraladministration, topical administration, parenteral administration,intravenous administration, subcutaneous administration, intra-articularadministration, intramuscular administration, intraperitonealadministration, dermal administration, transdermal administration, or acombination thereof

In some aspects, a method of treating a condition in a subject in needthereof comprises administering to the subject the peptide active agentconjugate of any of embodiment as described herein, the peptide of anyof any embodiment as described herein, or a pharmaceutical compositionof any embodiment as described herein. In some embodiments, the peptideactive agent conjugate, peptide, or pharmaceutical composition isadministered by inhalation, intranasally, orally, topically,parenterally, intravenously, subcutaneously, intra-articularly,intramuscularly administration, intraperitoneally, dermally,transdermally, or a combination thereof. In some embodiments, thepeptide active agent conjugate or the peptide homes, targets, ormigrates to cartilage of the subject following administration. In someembodiments, the condition is associated with cartilage. In someembodiments, the condition is associated with a joint. In someembodiments, the condition is an inflammation, a cancer, a degradation,a growth disturbance, genetic, a tear, an infection, a disease, or aninjury. In some embodiments, the condition is a chondrodystrophy. Insome embodiments, the condition is a traumatic rupture or detachment. Insome embodiments, the condition is a costochondritis. In someembodiments, the condition is a herniation. In some embodiments, thecondition is a polychondritis. In some embodiments, the condition is achordoma. In some embodiments, the condition is a type of arthritis. Insome embodiments, the type of arthritis is rheumatoid arthritis. In someembodiments, the type of arthritis is osteoarthritis. In someembodiments, the condition is achondroplasia. In some embodiments, thecondition is benign chondroma or malignant chondrosarcoma. In someembodiments, the condition is bursitis, tendinitis, gout, pseudogout, anarthropathy, psoriatic arthritis, ankylosing spondylitis, or aninfection. In some embodiments, the peptide active agent conjugate,peptide, or pharmaceutical composition is administered to treat theinjury, to repair a tissue damaged by the injury, or to treat a paincaused by the injury. In some embodiments, the peptide active agentconjugate, peptide, or pharmaceutical composition is administered totreat the tear or to repair a tissue damaged by the tear. In someembodiments, the peptide active agent conjugate, peptide, orpharmaceutical composition homes, targets, or migrates to a kidney ofthe subject following administration. In some embodiments, the conditionis associated with a kidney. In some embodiments, the condition is lupusnephritis, acute kidney injury (AKI), chronic kidney disease (CKD),hypertensive kidney damage, diabetic nephropathy, or renal fibrosis.

In some aspects, a method of imaging an organ or body region of asubject comprises: administering to the subject the peptide active agentconjugate of any embodiment as described herein, the peptide of anyembodiment as described herein, or the pharmaceutical composition of anyembodiment as described herein; and imaging the subject. In someembodiments, the method further comprises detecting a cancer or diseasedregion, tissue, structure, or cell. In some embodiments, the methodfurther comprises performing surgery on the subject. In someembodiments, the method further comprises treating the cancer. In someembodiments, the surgery comprises removing the cancer or the diseasedregion, tissue, structure, or cell of the subject. In some embodiments,the method further comprises imaging the cancer or diseased region,tissue, structure, or cell of the subject after surgical removal. Insome embodiments, the peptide active agent conjugate is expressed as afusion protein.

In various aspects, compounds of the present dislcosure have thestructure of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, C₁-C₆ alkoxy, C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀alkylene-(C (═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, —COOH, C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀alkylene-(C (═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆alkylene-(O—-C₁-C₆ alkylene)_(n)—, —NR¹⁰—C₁-C₆ alkylene-NR¹¹—(C (═O)—C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl-, or —heterocyclyl-C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are each independently selected from hydrogen, C₁-C₆ alkyl,or R¹² and R¹³ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-aryl-A⁵,-(L⁵)-heteroaryl, — (L⁵)-heteroaryl-A⁵, —NR¹⁷ R¹⁸, R¹⁴ and R¹⁹ arejoined together along with the other atoms to which they are attached toform a 5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴and R²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, or —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are each independently selected from hydrogen, C₁-C₆ alkyl,R¹⁴ and R¹⁹ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

one of A¹, A², A³, A⁴, or A⁵ is a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof and the others of A¹, A², A³, A⁴, or A⁵ are eachindependently absent, hydrogen, — COOH, or sulfonate.

In various aspects, the presently described compounds further comprise adetectable label, which can be used for the detection of thepeptide-label conjugate and the cancerous cells to which they are bound.

In various aspects, compounds of the present dislcosure have thestructure of Formula (XV), or a pharmaceutically acceptable saltthereof:

wherein:

R³, R⁴, R⁵, R⁶, R¹⁵, and R¹⁶ are each independently selected fromhydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate, —COOH, —SO₂—NH₂,C₁-C₆ alkoxy, C₁-C₁₀ alkylene-(C(═O))_(x)—, C₁-C₁₀ alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, —COOH, C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀alkylene-(C (═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—C₁-C₆alkylene-(O—-C₁-C₆ alkylene)_(n)—, —NR¹⁰—C₁-C₆ alkylene-NR¹⁰-C₁-C₆alkylene—NR¹¹—(C (═O) —C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl-, or -heterocyclyl-C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are each independently selected from hydrogen, C₁-C₆ alkyl,or R¹² and R¹³ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-aryl-A⁵,-(L⁵)-heteroaryl, -(L⁵)-heteroaryl-A⁵, —NR¹⁷ R¹⁸, R¹⁴ and R¹⁹ are joinedtogether along with the other atoms to which they are attached to form a5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴ andR²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, or —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are each independently selected from hydrogen, C₁-C₆ alkyl,R¹⁴ and R¹⁹ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

R²¹ and R²² are each independently selected from hydrogen, C₁-C₆ alkyl,sulfonate, or R²¹ and R²² are joined together along with the other atomsto which they are attached to form a 5-membered or 6-membered aryl;

R²³ and R²⁴ are each independently selected from hydrogen, C₁-C₆ alkyl,sulfonate, or R²³ and R²⁴ are joined together along with the other atomsto which they are attached to form a 5-membered or 6-membered aryl;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

one of A¹, A², A³, A⁴, or A⁵ is a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof and the others of A¹, A², A³, A⁴, or A⁵ are eachindependently absent, hydrogen, —COOH, or sulfonate.

In some aspects, the compounds of the present disclosure have astructure of Formula (II), or a pharmaceutically acceptable saltthereof:

In certain aspects, the present compounds have a structure of Formula(III), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R⁹ is hydrogen, sulfonate, or —COOH;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆alkylene- (O—C₁-C₆ alkylene)_(n)-, —NR¹⁰-L⁴-, —NR¹⁰—C₁-C₆alkylene-NR¹¹—(C (═O) —C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3; and

A⁴ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In other aspects, compounds of the present disclosure have a structureof Formula (IV), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, and R¹⁶ are each independently selectedfrom hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate, —COOH,—SO₂—NH₂, or C₁-C₆ alkoxy;

R³ is selected from C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, or —COOH, or C₁-C₁₀ alkyl;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is hydrogen, sulfonate, —COOH, C₁-C₁₀ alkyl;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

A¹ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In other aspects, compounds of the present disclosure have a structureof Formula (V), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R¹⁵, and R¹⁶ are each independently selectedfrom hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate, —COOH,—SO₂—NH₂, or C₁-C₆ alkoxy;

R⁵ is selected from C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, or —COOH, or C₁-C₁₀ alkyl;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is hydrogen, sulfonate, —COOH, or C₁-C₁₀ alkyl;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

A² is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In some aspects, compounds of the present disclosure have a structure ofFormula (VI), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R⁹ is selected from C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is hydrogen, sulfonate, —COOH, or C₁-C₁₀ alkyl;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl-C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

A³ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In additional aspects, compounds of the present disclosure have astructure Formula (III), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R⁹ is hydrogen, sulfonate, or —COOH;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆alkylene- (O—C₁-C₆ alkylene)_(n)—, —NR¹⁰-L⁴-, —NR¹⁰-C₁-C₆alkylene-NR¹¹—(C (═O)—C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl-C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is -(L⁵)-aryl-A⁵, or -(L⁵)-heteroaryl-A⁵;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1;

A⁴ is hydrogen, —COOH, or sulfonate; and

A⁵ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In various aspects, the present disclosure provides a kit comprisingvessel configured to contain a fluid; any of the compounds andcompositions described herein; and an elastomeric closure affixed to thevessel.

In various aspects, the present disclosure provides a compositioncomprising a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof, wherein when the composition is intravenouslyadministering to a human subject at a dose of from 1 mg to 30 mg, thecomposition produces in the human subject an average maximum compoundblood plasma concentration (average C_(max)) of at least from 110 ng/mLto240 ng/mL per each 1 mg dosage of the compound administered.

In various aspects, the present disclosure provides a method ofadministering a composition to a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof; and producing in the human subject an average maximumcompound blood plasma concentration (average C_(max))of at least from110 ng/mL to240 ng/mL per each 1 mg dosage of the compound administered.

In various aspects, the present disclosure provides a method ofdetecting a cancer cell in a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof conjugated to a detectable label; producing in thehuman subject an average maximum compound blood plasma concentration(average C_(max))of at least from 110 ng/mL to 240 ng/mL per each 1 mgdosage of the compound administered; and detecting the presence orabsence of the detectable label in the human subject, wherein thepresence of the detectable label indicates the presence of the cancercell.

In various aspects, the present disclosure provides a method ofdiagnosing cancer in a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof conjugated to a detectable label; producing in thehuman subject an average maximum compound blood plasma concentration(average C_(max))of at least from 110 ng/mL to240 ng/mL per each 1 mgdosage of the compound administered; and detecting the presence orabsence of the detectable label in the human subject, wherein thepresence of the detectable label indicates a diagnosis of cancer.

In various aspects, the present disclosure provides a method of treatingcancer in a human subject, the method comprising: intravenouslyadministering to the human subject a dose of from 1 mg to 30 mg of acompound comprising a polypeptide having at least 85% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereofconjugated to a therapeutic agent; producing in the human subject anaverage maximum compound blood plasma concentration (average C_(max)) ofat least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of thecompound administered; and reducing or improving a symptom or conditionassociated with cancer in the human subject. In some aspects, the humansubject is in need thereof In some aspects, the methods compriseadministering a therapeutically effective dose of the compound to thehuman subject.

In various aspects, the present disclosure provides a method ofadministering a composition to a human subject, the method comprising:administering to the human subject a dose of from 1 mg to 30 mg of acompound comprising a polypeptide having at least 85% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof; andproducing in the human subject pharmacokinetic profile of FIG. 27 .

In various aspects, the present disclosure provides a method ofadministering a composition to a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of any suitable compound of the present disclosure; and producingin the human subject an average maximum compound blood plasmaconcentration (average C_(max)) of at least from 110 ng/mL to240 ng/mLper each 1 mg dosage of the compound administered.

In various aspects, the present disclosure provides a method fordetecting a cancer cell in a subject, the method comprising:administering any suitable compound of the present disclosure; anddetecting the presence or absence of the compound in the subject,wherein the presence of the compound indicates the presence of a cancercell.

In various aspects, the present disclosure provides a method ofadministering any suitable compound of the present disclosure to asubject, the method comprising administering a therapeutically effectiveamount of the compound to the subject.

In various aspects, the present disclosure provides a method of treatinga subject in need thereof, the method comprising administering to thesubject any suitable compound of the present disclosure furthercomprising a therapeutic agent in an amount sufficient to treat cancerin the subject. In certain aspects, the therapeutic agent is a cytotoxicagent.

In one embodiment, the chlorotoxin conjugate comprises achemotherapeutic, an anti-cancer agent, or an anti-cancer drug. Inanother embodiment, the chlorotoxin conjugate comprising thechemotherapeutic, an anti-cancer agent, or an anti-cancer drug isadministered after the central or primary tumor is detected duringsurgery. In a further embodiment, the central or primary tumor isdetected with a chlorotoxin conjugated to a labeling agent.

In another aspect is provided a method of inhibiting, preventing,minimizing, shrinking, or killing cells, or preventing metastasis inresidual tumor cells in a tumor bed in an individual, comprising thestep of administering a chlorotoxin conjugate to the individual whereinthe chlorotoxin conjugate binds to the residual tumor cells in a tumorbed; and whereby the residual tumor cells in the tumor bed areinhibited, prevented, minimized, shrinked, or killed cells, ormetastasis is prevented. In one embodiment, the chlorotoxin conjugatecomprises a chemotherapeutic, an anti-cancer agent, or an anti-cancerdrug. In another embodiment, the chlorotoxin conjugate comprising thechemotherapeutic, an anti-cancer agent, or an anti-cancer drug isadministered after the central or primary tumor is detected duringsurgery. In a further embodiment, the central or primary tumor isdetected with a chlorotoxin conjugated to a labeling agent.

In another aspect the invention provides, a method of administering achlorotoxin conjugated to a chemotherapeutic, an anti-cancer agent, oran anti-cancer drug to an individual to treat, inhibit, prevent,minimize, shrink, or kill cells, or prevent metastasis in cells that areidentified with a chlorotoxin conjugated to a labeling agent. In someembodiments the anti-cancer agent include antibodies, polypeptides,polysaccharides, and nucleic acids. In an embodiment, the chlorotoxinconjugate is administered about 1 day before the surgery. In anotherembodiment, the chlorotoxin conjugate is administered about 2 daysbefore surgery. In another embodiment, the chlorotoxin conjugate isadministered in multiple sub-doses. In an embodiment, the chlorotoxinconjugate is administered in about 2 sub-doses, 3 sub-doses, 4sub-doses, or more sub-doses. In another embodiment, the chlorotoxinconjugate comprising the chemotherapeutic, an anti-cancer agent, or ananti-cancer drug is administered after the central or primary tumor isdetected during surgery. In a further embodiment, the central or primarytumor is detected with a chlorotoxin conjugated to a labeling agent.

In one aspect, the invention provides a method of detecting soft-tissuesarcoma in an individual, comprising the steps of: a) administering achlorotoxin conjugate to the individual wherein the chlorotoxinconjugate binds to the soft-tissue sarcoma; and b) imaging, visualizing,or analyzing the bound chlorotoxin conjugate. In an embodiment, thedetecting comprises in vivo, or ex vivo detecting. In anotherembodiment, the imaging, visualizing, or analyzing comprises visualizingthe chlorotoxin conjugate. In a further embodiment, the imaging,visualizing, or analyzing comprises in vivo, or ex vivo imaging,visualizing, or analyzing. In some embodiments, the visualizingcomprises optically imaging the sarcoma. In some embodiments a plot ismade of the fluorescent intensity of the chlorotoxin conjugate.

In an aspect, a chlorotoxin conjugate comprising one or more labelingagents is used in detecting the soft-tissue sarcoma. In an embodiment,the labeling agent comprises a fluorescent moiety. In a furtherembodiment, the fluorescent moiety comprises a near infrared fluorescentmoiety. In another embodiment, the labeling agent comprises aradionuclide. In another embodiment, the soft-tissue sarcoma is selectedfrom the group consisting of: trachea, fat tissue tumors, muscle tissuetumors, skeletal muscle sarcomas, rhabdomyosarcomas, peripheral nervetumors, fibrous tissue tumors, myxofibrosarcomas, fibromatosis, jointtissue tumors, tumors of blood vessels and lymph vessels, angiosarcomas,gastrointestinal stromal tumors, alveolar soft part sarcoma,dermatofibrosarcoma protuberans (DFSP), desmoplastic small round celltumour, epithelioid sarcoma, extra skeletal myxoid chondrosarcoma, andgiant cell fibroblastoma (GCF).

In another aspect a chlorotoxin conjugate is used to detect soft-tissuesarcoma in subcutaneous fatty tissue. In an embodiment, the detectingcomprises imaging, visualizing, or analyzing the chlorotoxin conjugateduring or related to surgery, surgical resection, or intraoperativeimaging and resection. In another embodiment, the sarcoma, or a portionthereof, is removed during or related to surgery.

The invention provides a method of detecting cutaneous squamous cellcarcinoma in an individual, comprising the steps of: a) administering achlorotoxin conjugate to the individual, wherein the chlorotoxinconjugate binds to the cutaneous squamous cell carcinoma; and b)imaging, visualizing, or analyzing the bound chlorotoxin conjugate. Inan embodiment, the detecting comprises in vivo, or ex vivo detection. Inanother embodiment, the imaging, visualizing, or analyzing comprisesvisualizing the chlorotoxin conjugate. In another embodiment, theimaging, visualizing, or analyzing comprises in vivo, or ex vivoimaging, visualizing, or analyzing.

In another aspect, the invention provides a method of using achlorotoxin conjugate to optically image cutaneous squamous cellcarcinoma. In an embodiment, the chlorotoxin conjugate comprises one ormore labeling agents. In another embodiment, the labeling agentcomprises a fluorescent moiety. In a further embodiment, the fluorescentmoiety comprises a near infrared fluorescent moiety. In anotherembodiment, the labeling agent comprises a radionuclide. In someembodiments, the detecting comprises imaging, visualizing, or analyzingthe chlorotoxin conjugate during or related to surgery, surgicalresection, or intraoperative imaging and resection. In some embodiments,the cutaneous squamous cell carcinoma, or a portion thereof, is removedduring or related to surgery. In some embodiments the fluorescentintensity of the chlorotoxin conjugate is made.

In an aspect the invention provides a method of detecting a low-gradetumor in an individual, comprising the steps of: a) administering achlorotoxin conjugate to the individual wherein the chlorotoxinconjugate binds to the low-grade tumor; and b) imaging, visualizing, oranalyzing the bound chlorotoxin conjugate. In an embodiment, thedetecting comprises in vivo, or ex vivo detecting. In some embodiments,the imaging, visualizing, or analyzing comprises vivo, or ex vivoimaging, visualizing, or analyzing.

The invention provides a method of using a chlorotoxin conjugate tooptically image a low-grade tumor. In an embodiment, the chlorotoxinconjugate comprises one or more labeling agents. In an embodiment, thelabeling agent comprises a fluorescent moiety. In an embodiment, thefluorescent moiety comprises a near infrared fluorescent moiety. Inanother embodiment, the labeling agent comprises a radionuclide. Inanother embodiment, the detecting is performed during or related tosurgery or resection. In a further embodiment, the low-grade tumor, or aportion thereof, is removed during or related to surgery, surgicalresection, or intraoperative imaging and resection. In some embodiments,the low-grade tumor is selected from the group consisting of: a) alow-grade tumor in or from brain tissue; b) a low-grade tumor in or fromsubcutaneous fatty tissue; c) a low-grade tumor in or from breast ormammary tissue, and d) a low-grade tumor in or from lung tissue. In someembodiments a plot is made of the fluorescent intensity of thechlorotoxin conjugate.

In an embodiment, the chlorotoxin conjugate comprises one or morelabeling agents. In an embodiment, the labeling agent comprises afluorescent moiety. In an embodiment, the fluorescent moiety comprises anear infrared fluorescent moiety. In another embodiment, the labelingagent comprises a radionuclide. In an embodiment, the detectingcomprises in vivo, or ex vivo detection. In an embodiment, the imaging,visualizing, or analyzing comprises in vivo, or ex vivo imaging,visualizing, or analyzing. In some embodiments a plot is made of thefluorescent intensity of the chlorotoxin conjugate.

In another aspect, is provided a method for using a chlorotoxinconjugate to detect residual cancer in the tumor bed of an individualfollowing removal of a primary or central tumor in breast cancersurgery, comprising: a) administering a chlorotoxin conjugate to theindividual wherein the chlorotoxin conjugate binds to residual cancer;and b) imaging, visualizing, or analyzing the bound chlorotoxinconjugate. In an embodiment, the chlorotoxin conjugate comprises one ormore labeling agents. In a further embodiment, the labeling agentcomprises a fluorescent moiety. In another embodiment, the fluorescentmoiety comprises a near infrared fluorescent moiety. In anotherembodiment, the labeling agent comprises a radionuclide. In someembodiments the detecting is performed during or related to surgery orresection. In further embodiments, the residual cancer, or a portionthereof, is removed during or related to surgery, surgical resection, orintraoperative imaging and resection.

In an embodiment, the chlorotoxin conjugate comprises one or morelabeling agents. In a further embodiment, the labeling agent comprises afluorescent moiety. In another embodiment, the fluorescent moietycomprises a near infrared fluorescent moiety. In another embodiment, thelabeling agent comprises a radionuclide. In an aspect, the inventionprovides, a method for detecting a tumor in an individual comprising thesteps of: a) administering a chlorotoxin conjugate to the individualwherein the chlorotoxin conjugate binds to the tumor; and b) imaging,visualizing, or analyzing the bound chlorotoxin conjugate wherein thechlorotoxin conjugate is administered in an amount of between about 0.9mg/m² to about 1.1 mg/m² or in an amount of between about 3 mg to about6 mg. In an embodiment, the detecting comprises vivo, or ex vivodetection. In some embodiments, the imaging, visualizing, or analyzingcomprises in vivo, or ex vivo imaging, visualizing, or analyzing.

The invention provides a method of using a chlorotoxin conjugate tooptically image a tumor, in an embodiment, the chlorotoxin conjugatecomprises one or more labeling agents. In a father embodiment, thelabeling agent comprises a fluorescent moiety. In a further embodiment,the fluorescent moiety comprises a near infrared fluorescent moiety. Inanother embodiment, the labeling agent comprises a radionuclide. In someembodiments the detecting is performed during or related to surgery orresection. In some embodiments, the tumor, or a portion thereof, isremoved during or related to surgery surgical resection, orintraoperative imaging and resection.

The invention provides methods of administering a chlorotoxin conjugateto an individual to detect soft-tissue sarcoma, low-grade tumor,cutaneous squamous cell carcinoma, or cells therefrom, in tumors of skinor breast, and lung and mammary cancers. In an embodiment, thechlorotoxin conjugate comprises one or more labeling agents. In anembodiment, the labeling agent comprises a fluorescent moiety. In anembodiment, the fluorescent moiety comprises a near infrared fluorescentmoiety. In another embodiment, the labeling agent comprises aradionuclide. In another embodiment, the detecting is performed duringor related to surgery or resection. In a further embodiment, thesoft-tissue sarcoma, low-grade tumor, cutaneous squamous cell carcinoma,or cells therefrom, in tumors of skin or breast, and lung and mammarycancers, or a portion thereof, is removed during or related to surgery,surgical resection, or intraoperative imaging and resection. In otherembodiments, the chlorotoxin conjugate is administered in an amount ofbetween about 0.9 mg/m² to about 1.1 mg/m² or in an amount of betweenabout 3 mg to about 6 mg. In an embodiment, the chlorotoxin conjugate isadministered about 1 day before the surgery. In another embodiment, thechlorotoxin conjugate is administered about 2 days before surgery. Inanother embodiment, the chlorotoxin conjugate is administered inmultiple sub-doses. In an embodiment, the chlorotoxin conjugate isadministered in about 2 sub-doses, 3 sub-doses, 4 sub-doses, or moresub-doses. In an embodiment, the detecting comprises in vivo, or ex vivodetection. In some embodiments, the imaging, visualizing, or analyzingcomprises in vivo, or ex vivo imaging, visualizing, or analyzing. Insome embodiments a plot is made of the fluorescent intensity of thechlorotoxin conjugate.

In another aspect, is provided a method for using a chlorotoxinconjugate to detect residual cancer in the tumor bed of an individualfollowing removal of a primary or central tumor in breast cancersurgery, comprising: a) administering a chlorotoxin conjugate to theindividual wherein the chlorotoxin conjugate binds to residual cancer;and b) imaging, visualizing, or analyzing the bound chlorotoxinconjugate. In an embodiment, the chlorotoxin conjugate comprises one ormore labeling agents. In a further embodiment, the labeling agentcomprises a fluorescent moiety. In another embodiment, the fluorescentmoiety comprises a near infrared fluorescent moiety. In anotherembodiment, the labeling agent comprises a radionuclide. In someembodiments the detecting is performed during or related to surgery orresection. In further embodiments, the residual cancer, or a portionthereof, is removed during or related to surgery, surgical resection, orintraoperative imaging and resection.

In an embodiment, the chlorotoxin conjugate comprises one or morelabeling agents. In an embodiment, the labeling agent comprises afluorescent moiety. In an embodiment, the fluorescent moiety comprises anear infrared fluorescent moiety. In another embodiment, the labelingagent comprises a radionuclide. In another embodiment, the detecting isperformed during or related to surgery or resection. In an embodiment,the chlorotoxin conjugate is administered about 1 day before thesurgery. In another embodiment, the chlorotoxin conjugate isadministered about 2 days before surgery. In another embodiment, thechlorotoxin conjugate is administered in multiple sub-doses. In anembodiment, the chlorotoxin conjugate is administered in about 2sub-doses, 3 sub-doses, 4 sub-doses, or more sub-doses. In anembodiment, the detecting comprises vivo, or ex vivo detection. In someembodiments, the imaging, visualizing, or analyzing comprises in vivo,or ex vivo imaging, visualizing, or analyzing. In some embodiments aplot is made of the fluorescent intensity of the chlorotoxin conjugate.

The invention further provides methods for detecting soft tissue sarcomain an individual comprising, administering a chlorotoxin conjugate tothe individual, wherein the chlorotoxin conjugate comprises a detectableagent and a chlorotoxin polypeptide having at least 85% sequenceidentity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, binding thechlorotoxin conjugate to the soft tissue sarcoma, and detecting thebound chlorotoxin conjugate, wherein an elevated level of boundchlorotoxin conjugate indicates the presence of soft tissue sarcoma.

Also described herein are peptides that selectively bind to cancercells. These peptides may be conjugated to a fluorescent moiety, such asan indocyanine green molecule, to form a peptide conjugate thatselectively binds to cancer cells. Methods of selectively labelingcancer cells may utilize a peptide conjugate comprising a tumor-homingpeptide and a fluorescent moiety.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A-FIG. 1C shows SDS-PAGE of chlorotoxin conjugate formulationsafter 14 days at room temperature.

FIG. 2A-FIG. 2C shows SDS-PAGE of chlorotoxin conjugate formulationsafter 5 days at 40° C_(max) (and t0).

FIG. 3A-FIG. 3C shows SDS-PAGE of chlorotoxin conjugate formulationsafter 3 days at room temperature in the light or 3X F/T. Samples areafter 3 days at room temperature in the light unless marked as F/T

FIG. 4 shows total peak area of RP-HPLC chromatograms for chlorotoxinconjugate formulations with no lyophilization or lyophilization withfast or slow freezing.

FIG. 5 shows FTIR-derived secondary structures obtained for chlorotoxinconjugate lyophilized with slow versus fast freezing (left) and in aliquid formulation (right).

FIG. 6 shows SDS-PAGE analysis of various lyophilized formulations.

FIG. 7 shows signal-to-noise ratios (SNRs) of chlorotoxin conjugates inU87 flank tumors 24 hours after injection.

FIG. 8 shows fluorescent images of Compound 76-2kD (left), Compound76-5kD (middle) and Compound 76-40kD (right).

FIG. 9A shows a fluorescent image of Compound 76-5kD. FIG. 9B shows anH&E stain of the tissue sample.

FIG. 10A-FIG. 10C shows biodistribution analysis of Compound 76-5kD.FIG. 10A shows integrated intensity for sections from liver, kidney,spleen, heart and brain. FIGS. 10B and 10C show representative imagesfrom liver and kidney, respectively.

FIG. 11 shows an Odyssey fluorescent image of Compound 76-5kD (top) andH&E stain (bottom) of heart tissue.

FIG. 12A SNR (tumor/muscle) for different doses of chlorotoxin conjugate(n=2-7; sample size is shown inside each column; error=standarddeviation).

FIG. 12B shows signal from a subset of tumor and muscle samples.Representative muscle and tumor images for the 6 nmol and 20 nmol dosegroups 1 and 3 days post injection are shown below the graph.

FIG. 13A-FIG. 13C shows biodistribution of select tissues using the IVISSpectrum imaging system. FIG. 13A represents tissues one day afterinjection. FIG. 13B shows tissues imaged three days post injection.(B—brain, H—heart, K—kidney, S—skin, L—liver). FIG. 13C showsfluorescent signal in tissues for the 2 nmol and 20 nmol groups one andthree days after injection.

FIG. 14 shows whole body live animal imaging of chlorotoxin conjugatefrom six hours to three days after injection.

FIG. 15A shows ex vivo imaging of chlorotoxin conjugate 1 day afterinjection using the IVIS Spectrum. FIG. 15B shows ex vivo imaging of thebrain from orthotopic mouse #2. FIG. 15C shows ex vivo Odyssey imagingof the brain and skull from orthotopic mouse #1.

FIG. 16 shows fluorescence intensity in gross tumors, grouped by tumortype. Regions of interest (ROI) were drawn on Odyssey scans of grosstumors. ROIs were the same size across the data set.

FIG. 17 shows ex vivo imaging of a canine soft tissue sarcoma. Patient13 was a 7-year-old female Standard Poodle who presented with asubcutaneous hemangiopericytoma, a type of soft tissue sarcoma. She wastreated with BLZ-100 at 0.94 mg/m², and surgery was performed 48 hourslater. White light (A, C) and Odyssey near-infrared (B, D) images areshown of gross tumor and adjacent normal fat (A, B) and uninvolved skin(C, D). Gross ratios of tumor to fat were 257:1 to 127:1, and grossratios of tumor to skin were 89:1 to 33:1.

FIG. 18 shows a determination of the chlorotoxin conjugate dose at whichtumor to background ratio is maximal. Sections of tumor and non-tumortissue were scored by a histopathologist who was blinded to thefluorescence data. Total fluorescence in 2×2 mm grid squares wasmeasured, and each square was designated tumor or non-tumor byoverlaying the fluorescence image with the H&E image scored by thehistopathologist. The average of tumor and non-tumor squares was used tocompute the tumor to background ratio (TBR), shown at right of each datapoint.

FIG. 19 shows a box & whiskers plot of gross tumor intensity forsoft-tissue sarcomas (all subtypes) and carcinomas (includingadenocarcinoma and squamous cell carcinoma). Tissues were labeled withBLZ-100.

FIG. 20 shows signal and image analysis of tumor versus normal corticaltissue in SmoA1 mouse brain.

FIG. 21 shows small foci of fluorescence that correspond to smallclusters of tumor cells highlighted in the H&E stained slide.

FIG. 22 shows the mean (SD) of serum BLZ-100 concentration vs. timeprofiles following a single intravenous bolus of 0.02 mg BLZ-100administered to female mice.

FIG. 23 shows the mean (SD) of serum BLZ-100 concentration vs. timeprofiles following a single intravenous bolus of 0.03 or 0.3 mg dose tomale rats.

FIG. 24 shows the individual serum BLZ-100 concentration vs. timeprofiles following a single intravenous bolus dose of 0.6 mg to malemonkeys.

FIG. 25 shows the mean (±SD) BLZ-100 serum concentrations (ng/mL)summarized by dose following a single intravenous bolus dose to male andfemale rats.

FIG. 26 shows the mean (±SD) BLZ-100 serum concentrations (ng/mL)summarized by dose following a single intravenous bolus dose to male andfemale monkeys.

FIG. 27 shows chlorotoxin conjugate serum concentration vs time forhuman subjects.

FIG. 28 shows intraoperative imaging of a soft-tissue sarcoma (patient19). (A) White light preoperative image of gross tumor showing ulceratedand grossly swollen peritumoral skin. (B) NIR image of tumor in situ.(C) Plot of fluorescence intensity along the line drawn through theimage in panel B. (D) NIR image of excised tumor. (E) Plot offluorescence intensity along the line drawn through the image in panelD. (F) NIR image of peritumoral skin, surrounding uninvolved skin, andtumor bed. Peritumoral skin is 6-fold more intense than uninvolved skin.There is no residual fluorescence in the tumor bed. Tissues were labeledwith BLZ-100.

FIG. 29 shows intraoperative imaging of a mammary carcinoma (patient22). (A) NIR image of fluorescence from the primary mass (arrow) imagedfrom the bottom of the resected tissue, taken immediately post-excision.This aspect had a margin of about 0.5 cm of normal tissue. Panels B(fluorescence) and C (overlay) show the primary mass from the skin side,after the skin was opened and a slice removed for further imaging. Thesmall fluorescent patches were originally part of the primary mass, butwere separated by the removal of the slice. Panels D and E show thegross appearance and fluorescence overlay of the pieces submitted forfurther imaging. The contrast between gross tumor and adjacent tissue isabout 2.5-fold. (F) Overlay image of the tumor bed, showing lack ofresidual fluorescence in the muscle wall. Tissues were labeled withBLZ-100.

FIG. 30 shows intraoperative imaging of a cutaneous squamous cellcarcinoma (patient 23). (A) White light preoperative image of tumorsite, showing grossly ulcerated and swollen peritumoral skin. Panels Band C show preoperative NIR fluorescence images of the tumor from thetop (B) and side (C). NIR fluorescence (D) and overlay (E) are shownfollowing removal of the tail. The mass at right is a section of centraltumor removed for further analysis. The skin is retracted, and theremaining gross tumor (arrow) is revealed. Fluorescence intensity issimilar in the central tumor and remaining gross tumor, whileperitumoral skin has somewhat lower fluorescence intensity (F). Tissueswere labeled with BLZ-100.

FIG. 31 shows intraoperative imaging of a thyroid carcinoma (patient20). (Top) White light images of excised lymph node, thyroid mass, andtumor bed. (Middle) NIR fluorescence signal overlaid on white lightimages. (Bottom) Monochrome NIR images. The lymph node and thyroid masseach have a section removed for further analysis, shown side-by-sidewith the bulk tissue.

FIG. 32 shows a box & whiskers plots of fluorescence intensity in gridsquares for each tissue section analyzed. T, tumor. NT, adjacentnon-tumor tissue. PS, peritumoral skin. S, uninvolved skin. For thecutaneous tumors, the NT consisted of underlying dermis, subcutaneousfat, and adjacent dermis/epidermis. Tissues were labeled with achlorotoxin conjugate compound.

FIG. 33 shows imaging of a brain tumor labeled with BLZ-100. (A) NIRfluorescence image of tumor in situ. T, tumor. M, nasal mucosa. B,normal brain. Panels B and C show fluorescence images of 30 micronsections from two of the tumor pieces. Panels D and E show H&E stains ofthe sections in B and C, respectively.

FIG. 34 illustrates the identification of the ¹⁴C signal in the jointand other cartilage of an animal treated with the peptide of SEQ ID NO:511.

FIG. 35 illustrates a three-dimensional structure and a line structureof a peptide of SEQ ID NO: 515.

FIG. 36 illustrates an exemplary architecture of constructs expressingsequences of SEQ ID NO: X, where X can be any one of peptides of SEQ IDNO: 508—SEQ ID NO: 520.

FIG. 37 illustrates a schematic of a method of manufacturing of apeptide of the disclosure.

FIG. 38A-FIG. 38C illustrates alignment of SEQ ID NO: 1025 (SEQ ID NO:1025 is SEQ ID NO: 511, but without the first three amino acids “GSG”and is also SEQ ID NO: 801, but without the first amino acid “G”) withSEQ ID NO: 800, SEQ ID NO: 1025 with SEQ ID NO: 1026 (SEQ ID NO: 1026 isSEQ ID NO: 514, but without the first three amino acids “GSQ” and is SEQID NO: 804, but without the first amino acid “Q”), and SEQ ID NO: 1025with SEQ ID NO: 967. FIG. 38A illustrates the alignment of the peptideof SEQ ID NO: 1025 with the peptide of SEQ ID NO: 800. Boxes delineateconserved positively charged residues. FIG. 38B illustrates thealignment of the peptide of SEQ ID NO: 1025 with the peptide of SEQ IDNO: 1026. Boxes delineate conserved positively charged residues. FIG.38C illustrates the alignment of the peptide of SEQ ID NO: 1025 with thepeptide of SEQ ID NO: 967. Boxes delineate conserved positively chargedresidues.

FIG. 39 illustrates the alignment of the peptide of SEQ ID NO: 804 withthe peptide of SEQ ID NO: 968. Boxes delineate conserved positivelycharged residues.

FIG. 40 illustrates alignment of peptides within the pfam00451:toxin 2structural class family of SEQ ID NO: 978—SEQ ID NO: 1024. Boxed andbolded residues indicate relative conservation of sequence whilenon-boxed and non-bolded residues indicate areas of higher sequencevariability.

FIG. 41 illustrates alignment of a peptide of SEQ ID NO: 978 from thepfam00451:toxin 2 structural class family with a cartilage homingpeptide of this disclosure of SEQ ID NO: 511. Asterisks indicatepositions with a single, fully conserved residue, a colon indicatesconservation between groups of strongly similar properties (scoring >0.5in the Gonnet point accepted mutation (PAM) 250 matrix), and a periodindicates conservation between groups of weakly similar properties(scoring ≤0.5 in the Gonnet PAM 250 matrix).

FIG. 42 illustrates the ¹⁴C signal in the cartilage of an animal withintact kidneys 24 hours after treatment with a peptide of SEQ ID NO:511.

FIG. 43A-FIG. 43F shows white light images and corresponding whole bodyfluorescence images of a mouse administered 10 nmol of a peptide of SEQID NO: 592 conjugated to a Cy5.5 fluorophore (SEQ ID NO: 592A) at 24hours post-administration. FIG. 43A illustrates an image of a frozensection of a mouse, 24 hours after administration of 10 nmol of apeptide of SEQ ID NO: 592 conjugated to a Cy5.5 fluorophore (SEQ ID NO:592A). FIG. 43B illustrates the fluorescence signal in the mouse,corresponding to the section shown in FIG. 43A, 24 hours afteradministration of 10 nmol of a peptide of SEQ ID NO: 592 conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A). FIG. 43C illustrates an image of adifferent frozen section of the mouse, 24 hours after administration of10 nmol of a peptide of SEQ ID NO: 592 conjugated to a Cy5.5 fluorophore(SEQ ID NO: 592A). FIG. 43D illustrates the fluorescence signal in themouse, corresponding to the section shown in FIG. 43C, 24 hours afteradministration of 10 nmol of a peptide of SEQ ID NO: 592 conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A). FIG. 43E illustrates an image of adifferent frozen section of the mouse, 24 hours after administration of10 nmol of a peptide of SEQ ID NO: 592 conjugated to a Cy5.5 fluorophore(SEQ ID NO: 592A). FIG. 43F illustrates a fluorescence signal in themouse, corresponding to the section shown in FIG. 43E, 24 hours afteradministration of 10 nmol of a peptide of SEQ ID NO: 592 conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A).

FIG. 44 illustrates a multiple sequence alignment of SEQ ID NO: 800, SEQID NO: 801, SEQ ID NO: 805, SEQ ID NO: 817, SEQ ID NO: 821, SEQ ID NO:822, SEQ ID NO: 824, SEQ ID NO: 882, SEQ ID NO: 958, SEQ ID NO: 967, SEQID NO: 970, and SEQ ID NO: 1027—SEQ ID NO: 1033 were used to predictenhanced peptide stability and immunogenicity. SEQ ID NO: 779 is aconsensus sequence.

FIG. 45 illustrates the identification of locations the ¹⁴C signal inthe nasal, spinal, tracheal, and other cartilage of an animal treatedwith the peptide of SEQ ID NO: 511.

FIG. 46A-FIG. 46H shows IVIS fluorescence imaging of an isolated hindlimb from a first mouse and an isolated hind limb from a second mouseafter administration of 10 nmol SEQ ID NO: 592 peptide conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A). Areas of low signal intensity areshown in a thin solid line, areas of medium signal intensity are shownin a thick sold line, and areas of high signal intensity are shown in athin dotted line. FIG. 46A shows the right hind limb with skin removedfrom a first mouse and from a second mouse 3 hours after peptideadministration. FIG. 46B shows the right hind limb with muscle removedfrom a first mouse and from a second mouse 3 hours after peptideadministration of 10 nmol SEQ ID NO: 592 peptide conjugated to a Cy5.5fluorophore (SEQ ID NO: 592A). FIG. 46C shows the right hind limb withskin removed from a first mouse and from a second mouse 24 hours afterpeptide administration of 10 nmol SEQ ID NO: 592 peptide conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A). FIG. 46D shows the right hind limbwith muscle removed from a first mouse and from a second mouse 24 hoursafter peptide administration of 10 nmol SEQ ID NO: 592 peptideconjugated to a Cy5.5 fluorophore (SEQ ID NO: 592A). FIG. 46E shows theright hind limb with skin removed from a first mouse and from a secondmouse 48 hours after peptide administration of 10 nmol SEQ ID NO: 592peptide conjugated to a Cy5.5 fluorophore (SEQ ID NO: 592A). FIG. 46Fshows the right hind limb with muscle removed from a first mouse andfrom a second mouse 48 hours after peptide administration of 10 nmol SEQID NO: 592 peptide conjugated to a Cy5.5 fluorophore (SEQ ID NO: 592A).FIG. 46G shows the right hind limb with skin removed from a first mouseand from a second mouse 72 hours after peptide administration of 10 nmolSEQ ID NO: 592 peptide conjugated to a Cy5.5 fluorophore (SEQ ID NO:592A). FIG. 46H shows the right hind limb with muscle removed from afirst mouse and from a second mouse 72 hours after administration of 10nmol SEQ ID NO: 592 peptide conjugated to a Cy5.5 fluorophore (SEQ IDNO: 592A).

FIG. 47A-FIG. 47B illustrates autoradiography images of frozen sectionsfrom a mouse, 3 hours after administration of 100 nmol of a radiolabeledpeptide of SEQ ID NO: 592. FIG. 47A illustrates the ¹⁴C signal in adifferent frozen section of the mouse, 3 hours after administration of100 nmol of a radiolabeled peptide of SEQ ID NO: 592. FIG. 47Billustrates the ¹⁴C signal in a different frozen section of a mouse, 3hours after administration of 100 nmol of a radiolabeled peptide of SEQID NO: 592.

FIG. 48A-FIG. 48B illustrates autoradiography images of frozen sectionsfrom a mouse, 3 hours after administration of 100 nmol of a radiolabeledpeptide of SEQ ID NO: 590. FIG. 48A illustrates the ¹⁴C signal in afrozen section of a mouse. FIG. 48B illustrates the ¹⁴C signal in afrozen section of the mouse, 3 hours after administration of 100 nmol ofa radiolabeled peptide of SEQ ID NO: 590.

FIG. 49A-FIG. 49B illustrates autoradiography images of frozen sectionsfrom a mouse, 3 hours after administration of 100 nmol of a radiolabeledpeptide of SEQ ID NO: 671.

FIG. 49A illustrates the ¹⁴C signal in a frozen section of the mouse, 3hours after administration of 100 nmol of a radiolabeled peptide of SEQID NO: 671. FIG. 49B illustrates the ¹⁴C signal in a frozen section ofthe mouse, 3 hours after administration of 100 nmol of a radiolabeledpeptide of SEQ ID NO: 671.

DETAILED DESCRIPTION

The present disclosure relates generally to compositions and methods forcartilage therapy. In some embodiments, the compositions and methodsherein utilize peptides that home, target, are directed to, are retainedby, accumulate in, migrate to, and/or bind to cartilage followingadministration to a subject. In some embodiments, the cartilage homingpeptides of the present disclosure exert therapeutic effect in cartilageor tissue or cell thereof. In some embodiments, the cartilage homingpeptides of the present disclosure are used to deliver an active agentto cartilage or tissue or cell thereof. The active agent can exert atherapeutic effect on cartilage or tissue or cell thereof. For example,in certain embodiments, the peptide itself or the active agent allowsfor localized delivery of an anti-inflammatory or other agent tocartilage or tissue or cell thereof. As another example, the activeagent is a fluorophore that can be used for imaging of cartilage. Incertain embodiments, the peptide itself induces therapeutic responses.

Cartilage disorders are particularly difficult to treat. A direct routefor active agent administration can be parenterally (e.g.,intravenously, subcutaneously, intramuscularly), intra-articularly, byinhalation, dermally, topically, or orally. However, cartilage can beavascular thus intravenous administration of drugs can fail to reach thecartilage in significant amounts. Drugs for cartilage diseases, such asosteoarthritis, can be injected directly locally into the affected area,for example, directly injected into the joint. Few drugs aimed attreating cartilage disorders have proved therapeutically viable withlack of access to target tissue being a primary reason for failure. Thelack of access to the target tissue can also lead to administration ofdoses that are higher than would be necessary if a drug could home,target, or be directed to, is retained by, and/or binds to a targetregion, tissue, structure or cell. Thus, treatment of cartilageconditions often requires the use of high concentrations of non-specificdrugs. In addition, a number of therapeutics are of interest in treatingjoint disorders, but are problematic because of the level of sideeffects caused by systemic administration of the drug (Dancevic andMcCulloch, Arthritis Res Ther. 16:429 (2014)).

Specific and potent drugs that are capable of contacting the cartilagecan counteract the non-specificity of many treatments by selectivelytargeting and delivering compounds to specific regions, tissues, cellsand structures. Such drugs can also be useful to modulate ion channels,protein-protein interactions, extracellular matrix remodeling (i.e.,protease inhibition), and the like. Such targeted therapy can allow forlower dosing, reduced side effects, improved patient compliance, andimprovement in therapeutic outcomes, which would be advantageous notonly in acute disease of the cartilage, but in chronic conditions aswell.

The present disclosure provides peptides that can comprise or can bederived from cystine-dense peptides. As used herein, the term“cystine-dense peptide” can be interchangeable with the terms “knottedpeptide,” “knottin,” and “optide,” and cystine-dense peptides can alsobe abbreviated as “CDPs.” Hitchins, amongst other disulfide-containingpeptides, can also be considered “knotted peptides” or “cystine-densepeptides” for the purposes of this disclosure. Knottins, for example,are a class of cystine-dense peptides comprising from about 11 to about80 amino acids in length that are often folded into a compact structure.Knottins and other cystine-dense peptides are typically assembled into acomplex tertiary structure that is characterized by a number ofintramolecular disulfide crosslinks and can contain beta strands, analpha helix, and other secondary structures. The presence of thedisulfide bonds can give cystine-dense peptides remarkable environmentalstability, allowing them to withstand extremes of temperature and pH, toresist proteolytic enzymes in the blood stream or digestive tract, andcan provide specific biodistribution, pharmacokinetic, bindinginteractions, cellular processing, or other properties of physiologicand therapeutic value. The peptides disclosed herein can be derived fromcertain cystine-dense peptides. The present disclosure describes a classof cystine-dense peptides that can effectively contact cartilage and beused either directly or as carriers of active drugs, peptides, ormolecules to treat a cartilage condition. For instance, osteoarthritisis a cartilage condition that is associated with the thinning ofcartilage covering the ends of bones resulting in bone directlycontacting bone within the joint. Over time, the ends of the bones aresubjected to increased levels of friction which ultimately causeserosion of the end of the bone. Individuals suffering fromosteoarthritis experience reduced motion and increased pain. Atherapeutic peptide that could contact the cartilage at the joint andends of the bone to interact with the chondrocytes and induce increasedexpression of extracellular matrix proteins could be used in thetreatment and prevention of osteoarthritis by increasing expression ofcollagen through, for example, the rate of production, amount ofproduction, inhibition of proteins which degrade collagen, promoteexpression of other proteins which maintain the integrity of existingcollagen proteins, or other mechanism. A peptide could also affectnearby tissues or cells such as the bone, ligaments, muscle, tendons,bursa, connective tissue, blood vessels, peripheral nerves, osteoclasts,osteoblasts, fibroblasts, synoviocytes, monocytes/macrophages,lymphocytes, plasma cells, adipocytes, endothelial cells, neurons,ligaments, muscle, tendons, and bursa. The peptides of the disclosurecan be used to treat the symptoms of various conditions. The peptides ofthe disclosure can bind to, home to, migrate to, accumulate in, beretained by, or be directed to cartilage and its components, includingchondrocytes, extracellular matrix, collagen, hyaluranon, aggrecan (alsoknown as cartilage-specific proteoglycan core protein (CSPCP)), or othercomponents of the extracellular matrix and the joint, or to other nearbycomponents such as those described herein in joints and cartilaginoustissues as listed above.

Also described herein are peptides that selectively home, target, aredirected to, migrate to, are retained by, or accumulate in and/or bindto specific regions, tissues, structures or cells of the cartilage thataid in managing, decreasing, ablating or reducing pain (e.g., jointpain) due to chronic disease or cartilage injury or other therapeuticindications as described herein. A peptide that homes, targets, migratesto, is directed to, is retained by, or accumulates in and/or binds toone or more specific regions, tissues, structures or cells of thecartilage can have fewer off-target and potentially negative effects,for example, side effects that often limit use and efficacy of paindrugs. In addition, such peptides can reduce dosage and increase theefficacy of existing drugs by directly targeting them to a specificregion, tissue, structure or cell of the cartilage and helping thecontact the cartilage or increasing the local concentration of agent.The peptide itself can modulate pain or it can be conjugated to an agentthat modulates pain. Such pain modulation may operate by variousmechanisms such as modulating inflammation, autoimmune responses, director indirect action on pain receptors, cell killing, or programmed celldeath (whether via an apoptotic and/or non-apoptotic pathway of diseasedcells or tissues, and the like (Tait et al., J Cell Sci 127(Pt10):2135-44 (2014)).

Peptides of this disclosure that home, target, are directed to, migrateto, are retained by, accumulate in, or bind to specific regions,tissues, structures or cells of the cartilage can do so with differentdegrees of efficiency. Peptides can have a higher concentration incartilage than in other locations, such as blood or muscle. Peptides canbe recorded as having a signal in cartilage as a percentage of signal inblood. For example, a cartilage signal of 200% indicates that the signalin cartilage is twice as high as the signal in blood. In someembodiments, peptides that have cartilage homing properties can have acartilage signal of >170% by radiographic densitometry measurements. Inother embodiments, peptides that are cartilage homers can have acartilage signal of >200% by radiographic densitometry measurements. Inother embodiments, peptides that are more efficient cartilage homers canhave a cartilage signal of >300% by radiographic densitometrymeasurements. In other embodiments, peptides that are more efficientcartilage homers can have a cartilage signal of >400% by radiographicdensitometry measurements. In other embodiments, peptides that arestrongest cartilage homers of highest interest can have a cartilagesignal of >500% by radiographic densitometry measurements. In someembodiments, measurement of the ratio of peptide concentration in blood,muscle, or other tissues relative to the peptide concentration incartilage can be performed using various methods including measuring thedensitometry signal of peptides labeled with radioisotopes (as describedabove), or by using other assays.

Peptides that selectively home, target, are directed to, migrate to, areretained by, or accumulate in and/or bind to specific regions, tissues,structures or cells of the cartilage can occur after administration ofthe peptide to a subject. A subject can be a human or a non-humananimal.

The peptides disclosed herein can be used as active agents, orconjugated to detection agents such a fluorophores, iodide-containingX-ray contrast agents, lanthanide chelates (e.g., gadolinium for MRIimaging), perfluorocarbons (for ultrasound), or PET tracers (e.g., 18For 11C) for imaging and tracing the peptide, or conjugated to agentssuch as anti-inflammatory active agents or other active agents to thejoint to treat inflammation or other disease.

The peptides disclosed herein can be used to bind cartilage explants exvivo. Cartilage explants can be from any subject, such as a human or ananimal. Assessment of peptide binding to cartilage explants can be usedto screen peptides that may efficiently home to cartilage in vivo

In some embodiments, peptides of this disclosure home, target, aredirected to, migrate to, are retained by, accumulate in, or bind tospecific regions, tissues, structures or cells of the kidneys. Forexample, in some embodiments, peptides of this disclosure home, target,are directed to, migrate to, are retained by, accumulate in, or bind tothe proximal tubules of the kidneys, kidney nephrons, or podocytes.Peptides that selectively home, target, are directed to, migrate to, areretained by, or accumulate in and/or bind to specific regions, tissues,structures or cells of the kidney can occur after administration of thepeptide to a subject. A subject can be a human or a non-human animal.The peptides disclosed herein can be used as active agents, orconjugated to detection agents such a fluorophores, iodide-containingX-ray contrast agents, lanthanide chelates (e.g., gadolinium for MRIimaging), perfluorocarbons (for ultrasound), or PET tracers (e.g., 18For 11C) for imaging and tracing the peptide, or conjugated to agentssuch as anti-inflammatory agents or other agents to the kidney to treatrenal cancer, chronic kidney failure or other kidney disease.

One roadblock in the advancement and wide spread use of peptides as atherapeutic is that peptides can be chemically and physically unstable.During the process of manufacturing of therapeutic peptides essentialconsiderations can include storage conditions, sustained biochemicalfunction, and in vivo delivery. Peptide degradation products can resultin the formation of species that alter the safety profile, potency, andimmunogenicity of the peptide. These peptide degradation products canform during manufacture and storage, as well as in vivo after deliveryto a patient. Furthermore, peptide degradation may limit the shelf-lifeand increase production cost due to unstable peptides requiringrefrigeration or shipment on dry ice. The latter can necessitatecontinual monitoring and validation of peptides as degradation productscould have formed during the manufacturing process. Hence, there is anurgent need for the rationale design and production of therapeuticpeptides that have enhanced stability, for example, in the ambientenvironment, during the process of manufacturing, in storage, and thatprevent the likelihood of peptide degradation under a variety ofconditions.

In some embodiments, the peptides and peptide-drug conjugates of thepresent disclosure have stability properties that minimize peptide orpeptide-drug conjugate degradation to enable adequate storage. Longterm, accelerated, and intermediate storage conditions for the peptidesand peptide-drug conjugates of the present disclosure can include longterm storage conditions of 25° C.±2° C./60% relative humidity (RH)±5%RH, or 30° C.±2° C./65% RH±5% RH for at least 6 months, at least 12months, and up to 1 year, up to 2 years, up to 3 years, up to 4 years,or longer than 4 years. In addition, intermediate and short term storageconditions (e.g., during transport, distribution, manufacturing, orhandling), or long term storage conditions for certain climates andinfrastructures, can include storage conditions of 30° C.±2° C./65%RH±5% RH or 40° C.±2° C./75% RH±5% RH for up to 1 hour, for up to 8hours, for up to 1 day, for up to 3 days, for up to 1 week, for up to 1month, for up to 3 months, for up to 6 months or at least 6 months, upto 1 year, up to 2 years, up to 3 years, up to 4 years, or longer than 4years). Moreover, the peptides and peptide-drug conjugates of thepresent disclosure can be refrigerated, for example between 5° C.±3° C.for at least 6 months, at least 12 months, and up to 1 year, up to 2years, up to 3 years, up to 4 years, or longer than 4 years. Inaddition, intermediate and short term refrigeration conditions (e.g.,during transport, distribution, manufacturing, or handling) can include25° C.±2° C./60% RH±5% RH for up to 1 hour, for up to 8 hours, for up to1 day, for up to 3 days, for up to 1 week, for up to 1 month, for up to3 months, for up to 6 months or at least 6 months, and potentiallylonger (at least 12 months and up to 1 year, up to 2 years, up to 3years, up to 4 years, or longer than 4 years). Such conditions forstorage, whether based on ambient or refrigerated conditions can beadjusted based upon the four zones in the world (e.g., the InternationalCouncil for Harmonisation of Technical Requirements for Pharmaceuticalsfor Human Use (ICH) stability Zone I, II, III, or IV) that aredistinguished by their characteristic prevalent annual climaticconditions. In addition, formulation components can be principallychosen for their ability to preserve the native conformation andchemical structure of the peptides and peptide-drug conjugates of thepresent disclosure in storage by preventing denaturation due tohydrophobic interactions and aggregation, as well as by preventingchemical degradation, including truncation, oxidation, deamidation,cleavage, hydrolysis, isomerization, disulfide exchange, racemization,and beta elimination (Cleland, et al., Crit Rev Ther Drug Carrier Syst10(4): 307-377 (1993); Shire et al., JPharm Sci 93(6): 1390-1402 (2004);Wakankar and Borchardt, JPharm Sci 95(11): 2321-2336 (2006)).

In some embodiments, the peptides and peptide-drug conjugates of thepresent disclosure have incorporated properties that minimizeimmunogenicity of the peptides and peptide-drug conjugates.Immunogenicity can be a major concern with the development oftherapeutic peptides and proteins, and there is an urgent need for therationale design and production of therapeutic peptides that havereduced immunogenicity and that increase their safety and efficacy.Immunogenicity can occur against a desired peptide sequence or a peptidedegradation product. Immunogenicity can occur when a patient develops animmune response to the therapeutic peptide, protein, conjugate, or otherdrug, such as by producing antibodies that bind to and/or neutralize thetherapeutic peptide, protein, conjugate, or other drug. The likelihoodof immunogenicity can increase when drugs are administered more thanonce or chronically. Immunogenicity can reduce patient exposure to thedrug, can reduce effectiveness of the drug, and can also result insafety risks for the patient, such as generating an immune response toself-proteins or other adverse responses related to increasedimmunogenicity to the therapeutic peptide, protein, conjugate, or otherdrug. Immunogenic responses can vary from patient to patient and alsoamongst different groups of HLA alleles, as well as over time. As such,minimizing risk of immunogenicity with a therapeutic peptide or proteincan be important for developing a drug that can be effectively andsafely used for treatment. Various methods exist for assessment ofimmunogenic potential, which can include in silico methods, in vitrotesting, preclinical in vivo testing, and assessment during clinicaldosing. Evaluation early in product design and development of thetherapeutic peptides and peptide-drug conjugates of the presentdisclosure in the in vivo milieu in which they function (e.g., ininflammatory environments or at physiologic pH) can revealsusceptibilities to modifications (e.g., aggregation and deamidation)that can result in loss of efficacy or induction of immune responses.Such information can be used to facilitate product engineering toenhance the stability of the product under such in vivo conditions orreduce immunogenicity. Moreover, the therapeutic peptides andpeptide-drug conjugates of the present disclosure can be designed tominimize protein aggregation. Strategies to minimize aggregate formationcan be used early in drug development, for example, by using anappropriate cell substrate, selecting manufacturing conditions thatminimize aggregate formation, employing a robust purification schemethat removes aggregates to the greatest extent possible, and choosing aformulation and container closure system that minimize aggregationduring storage.

Additional aspects and advantages of the present disclosure will becomeapparent to those skilled in this art from the following detaileddescription, wherein illustrative embodiments of the present disclosureare shown and described. As will be realized, the present disclosure iscapable of other and different embodiments, and its several details arecapable of modifications in various respects, all without departing fromthe disclosure. Accordingly, the drawings and description are to beregarded as illustrative in nature, and not as restrictive.

As used herein, the abbreviations for the natural L-enantiomeric aminoacids are conventional and are as follows: alanine (A, Ala); arginine(R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C,Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly);histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K,Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro);serine (S, Ser); threonine (T, Thr); tryptophan (R^(a), Trp); tyrosine(Y, Tyr); valine (V, Val). Typically, Xaa can indicate any amino acid.In some embodiments, X can be asparagine (N), glutamine (Q), histidine(H), lysine (K), or arginine (R).

Some embodiments of the disclosure contemplate D-amino acid residues ofany standard or non-standard amino acid or analogue thereof. When anamino acid sequence is represented as a series of three-letter orone-letter amino acid abbreviations, the left-hand direction is theamino terminal direction and the right-hand direction is the carboxylterminal direction, in accordance with standard usage and convention.

The present disclosure also provides compositions and methods for thedetection and/or treatment of cancers. The compositions described hereincomprise peptide conjugates comprising a detectable label, which aresuitable for the detection and treatment of various cancers. In certainaspects, the compositions are provided in combination with apharmaceutically acceptable carrier, which can be administered to asubject by any parenteral route of administration. The compositionsdescribed herein give rise to a pharmacokinetic profile whenadministered intravenously to a human subject. Following administrationof the compositions described herein, the conjugates bind selectively tocancer cells. The cancer cells can then be detected, for example, byimaging or other visualization or detection method suitable fordetecting the detectable label of the peptide conjugate. In furtheraspects, the presently described compositions can be used to treatcancer by way of a therapeutic agent, which is attached to the conjugateand which acts on the cancer cells following binding by the peptideportion of the conjugate. These and other aspects are described indetail herein.

The invention will best be understood by reference to the followingdetailed description of the aspects and embodiments of the invention,taken in conjunction with the accompanying drawings and figures. Thediscussion below is descriptive, illustrative and exemplary and is notto be taken as limiting the scope defined by any appended claims.

As used in the specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes a plurality of such compounds, reference to “an agent” includesa plurality of such agents, and reference to “the cell” includesreference to one or more cells (or to a plurality of cells) andequivalents thereof known to those skilled in the art, and so forth.When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary between 1% and 15% of the stated number or numericalrange. The term “comprising” (and related terms such as “comprise” or“comprises” or “having” or “including”) is not intended to exclude thatin other certain embodiments, for example, an embodiment of anycomposition of matter, composition, method, or process, or the like,described herein, may “consist of” or “consist essentially of” thedescribed features.

“Cyano” refers to the —CN radical.

“Nitro” refers to the —NO₂ radical.

“Oxa” refers to the —O— radical.

“Oxo” refers to the ═O radical.

“Thioxo” refers to the ═S radical.

“Imino” refers to the ═N—H radical.

“Hydrazino” refers to the ═N—NH₂ radical.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to fifteen carbon atoms (e.g., C₁—C₁₅alkyl). In certain embodiments, an alkyl comprises one to thirteencarbon atoms (e.g., C₁—C₁₃ kyl). In certain embodiments, an alkylcomprises one to eight carbon atoms (e.g., C₁-C₈ alkyl). In otherembodiments, an alkyl comprises five to fifteen carbon atoms (e.g.,C₅—C₁₅ alkyl). In other embodiments, an alkyl comprises five to eightcarbon atoms (e.g., C₅—C₈ alkyl). The alkyl is attached to the rest ofthe molecule by a single bond, for example, methyl (Me), ethyl (Et),n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.Unless stated otherwise specifically in the specification, an alkylgroup is optionally substituted by one or more of the followingsubstituents: halo, cyano, nitro, oxo, thioxo, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) isindependently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one double bond, and having from two to twelve carbon atoms. Incertain embodiments, an alkenyl comprises two to eight carbon atoms. Inother embodiments, an alkenyl comprises two to four carbon atoms. Thealkenyl is attached to the rest of the molecule by a single bond, forexample, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl,pent-1-enyl, penta-1,4-dienyl, and the like. Unless stated otherwisespecifically in the specification, an alkenyl group is optionallysubstituted by one or more of the following substituents: halo, cyano,nitro, oxo, thioxo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where tis 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂(where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl,fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl.

“Alkynyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one triple bond, having from two to twelve carbon atoms. Incertain embodiments, an alkynyl comprises two to eight carbon atoms. Inother embodiments, an alkynyl has two to four carbon atoms. The alkynylis attached to the rest of the molecule by a single bond, for example,ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unlessstated otherwise specifically in the specification, an alkynyl group isoptionally substituted by one or more of the following substituents:halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, —OR^(a), —SR^(a),—OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where tis 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂(where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl,fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, containing no unsaturation andhaving from one to twelve carbon atoms, for example, methylene,ethylene, propylene, n-butylene, and the like. The alkylene chain isattached to the rest of the molecule through a single bond and to theradical group through a single bond. The points of attachment of thealkylene chain to the rest of the molecule and to the radical group canbe through one carbon in the alkylene chain or through any two carbonswithin the chain. Unless stated otherwise specifically in thespecification, an alkylene chain is optionally substituted by one ormore of the following substituents: halo, cyano, nitro, aryl,cycloalkyl, heterocyclyl, heteroaryl, oxo, thioxo, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) isindependently hydrogen, alkyl, fluoroalkyl, carbocyclyl,carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl or heteroarylalkyl.

“Alkenylene” or “alkenylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onedouble bond and having from two to twelve carbon atoms, for example,ethenylene, propenylene, n-butenylene, and the like. The alkenylenechain is attached to the rest of the molecule through a double bond or asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkenylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, an alkenylene chain is optionally substituted by oneor more of the following substituents: halo, cyano, nitro, aryl,cycloalkyl, heterocyclyl, heteroaryl, oxo, thioxo, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) isindependently hydrogen, alkyl, fluoroalkyl, cycloalkyl, cycloalkylalkyl,aryl (optionally substituted with one or more halo groups), aralkyl,heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, andwhere each of the above substituents is unsubstituted unless otherwiseindicated.

“Aryl” refers to a radical derived from an aromatic monocyclic ormulticyclic hydrocarbon ring system by removing a hydrogen atom from aring carbon atom. The aromatic monocyclic or multicyclic hydrocarbonring system contains only hydrogen and carbon from six to eighteencarbon atoms, where at least one of the rings in the ring system isfully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system in accordance with the Hückel theory. Aryl groupsinclude, but are not limited to, groups such as phenyl, fluorenyl, andnaphthyl. Unless stated otherwise specifically in the specification, theterm “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant toinclude aryl radicals optionally substituted by one or more substituentsindependently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl,cyano, nitro, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted aralkenyl, optionally substitutedaralkynyl, optionally substituted carbocyclyl, optionally substitutedcarbocyclylalkyl, optionally substituted heterocyclyl, optionallysubstituted heterocyclylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, —R^(b)—OR^(a),—R^(b)—OC(O)—R^(a), —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(n)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂ , —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a)(where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2),where each R^(a) is independently hydrogen, alkyl, fluoroalkyl,cycloalkyl, cycloalkylalkyl, aryl (optionally substituted with one ormore halo groups), aralkyl, heterocyclyl, heterocyclylalkyl, heteroarylor heteroarylalkyl, each R^(b) is independently a direct bond or astraight or branched alkylene or alkenylene chain, and R^(c) is astraight or branched alkylene or alkenylene chain, and where each of theabove substituents is unsubstituted unless otherwise indicated.

“Aralkyl” refers to a radical of the formula —R^(c)-aryl where R^(c) isan alkylene chain as defined above, for example, benzyl, diphenylmethyland the like. The alkylene chain part of the aralkyl radical isoptionally substituted as described above for an alkylene chain. Thearyl part of the aralkyl radical is optionally substituted as describedabove for an aryl group.

“Aralkenyl” refers to a radical of the formula —R^(d)-aryl where R^(d)is an alkenylene chain as defined above. The aryl part of the aralkenylradical is optionally substituted as described above for an aryl group.The alkenylene chain part of the aralkenyl radical is optionallysubstituted as defined above for an alkenylene group.

“Aralkynyl” refers to a radical of the formula —R^(e)-aryl, where R^(e)is an alkynylene chain as defined above. The aryl part of the aralkynylradical is optionally substituted as described above for an aryl group.The alkynylene chain part of the aralkynyl radical is optionallysubstituted as defined above for an alkynylene chain.

“Carbocyclyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,which may include fused or bridged ring systems, having from three tofifteen carbon atoms. In certain embodiments, a carbocyclyl comprisesthree to ten carbon atoms. In other embodiments, a carbocyclyl comprisesfive to seven carbon atoms. The carbocyclyl is attached to the rest ofthe molecule by a single bond. Carbocyclyl may be saturated, (i.e.,containing single C—C bonds only) or unsaturated (i.e., containing oneor more double bonds or triple bonds.) A fully saturated carbocyclylradical is also referred to as “cycloalkyl.” Examples of monocycliccycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated carbocyclyl isalso referred to as “cycloalkenyl.” Examples of monocyclic cycloalkenylsinclude, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, andcyclooctenyl. Polycyclic carbocyclyl radicals include, for example,adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl,decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unlessotherwise stated specifically in the specification, the term“carbocyclyl” is meant to include carbocyclyl radicals that areoptionally substituted by one or more substituents independentlyselected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo,cyano, nitro, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted aralkenyl, optionally substitutedaralkynyl, optionally substituted carbocyclyl, optionally substitutedcarbocyclylalkyl, optionally substituted heterocyclyl, optionallysubstituted heterocyclylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, —R^(b)—OR^(a), —R^(b)—SR^(a),—R^(b)—OC(O)—R^(a), —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a)(where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2),where each R^(a) is independently hydrogen, alkyl, fluoroalkyl,cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl, each R^(b) isindependently a direct bond or a straight or branched alkylene oralkenylene chain, and R^(c) is a straight or branched alkylene oralkenylene chain, and where each of the above substituents isunsubstituted unless otherwise indicated.

“Carbocyclylalkyl” refers to a radical of the formula —R^(c)-carbocyclylwhere R^(c) is an alkylene chain as defined above. The alkylene chainand the carbocyclyl radical is optionally substituted as defined above.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodosubstituents.

“Fluoroalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more fluoro radicals, as defined above, forexample, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of thefluoroalkyl radical is optionally substituted as defined above for analkyl group.

“Heterocyclyl” refers to a stable 3- to 18-membered non-aromatic ringradical that comprises two to twelve carbon atoms and from one to sixheteroatoms selected from nitrogen, oxygen and sulfur. Unless statedotherwise specifically in the specification, the heterocyclyl radical isa monocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems. The heteroatoms in theheterocyclyl radical may be optionally oxidized. One or more nitrogenatoms, if present, are optionally quaternized. The heterocyclyl radicalis partially or fully saturated. The heterocyclyl may be attached to therest of the molecule through any atom of the ring(s). Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, the term “heterocyclyl” is meant to include heterocyclylradicals as defined above that are optionally substituted by one or moresubstituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl,oxo, thioxo, cyano, nitro, optionally substituted aryl, optionallysubstituted aralkyl, optionally substituted aralkenyl, optionallysubstituted aralkynyl, optionally substituted carbocyclyl, optionallysubstituted carbocyclylalkyl, optionally substituted heterocyclyl,optionally substituted heterocyclylalkyl, optionally substitutedheteroaryl, optionally substituted heteroarylalkyl, —R^(b)—OR^(a),—R^(b)—SR^(a), —R^(b)—OC(O) R^(a), —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a)(where t is 1 or 2) and -—^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2),where each R^(a). is independently hydrogen, alkyl, fluoroalkyl,cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl, each R^(b) isindependently a direct bond or a straight or branched alkylene oralkenylene chain, and R^(c) is a straight or branched alkylene oralkenylene chain, and where each of the above substituents isunsubstituted unless otherwise indicated.

“N-heterocyclyl” or “N-attached heterocyclyl” refers to a heterocyclylradical as defined above containing at least one nitrogen and where thepoint of attachment of the heterocyclyl radical to the rest of themolecule is through a nitrogen atom in the heterocyclyl radical. AnN-heterocyclyl radical is optionally substituted as described above forheterocyclyl radicals. Examples of such N-heterocyclyl radicals include,but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl,1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

“C-heterocyclyl” or “C-attached heterocyclyl” refers to a heterocyclylradical as defined above containing at least one heteroatom and wherethe point of attachment of the heterocyclyl radical to the rest of themolecule is through a carbon atom in the heterocyclyl radical. AC-heterocyclyl radical is optionally substituted as described above forheterocyclyl radicals. Examples of such C-heterocyclyl radicals include,but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl,2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.

“Heterocyclylalkyl” refers to a radical of the formula—R^(c)-heterocyclyl where R^(c) is an alkylene chain as defined above.If the heterocyclyl is a nitrogen-containing heterocyclyl, theheterocyclyl is optionally attached to the alkyl radical at the nitrogenatom. The alkylene chain of the heterocyclylalkyl radical is optionallysubstituted as defined above for an alkylene chain. The heterocyclylpart of the heterocyclylalkyl radical is optionally substituted asdefined above for a heterocyclyl group.

“Heteroaryl” refers to a radical derived from a 3- to 18-memberedaromatic ring radical that comprises two to seventeen carbon atoms andfrom one to six heteroatoms selected from nitrogen, oxygen and sulfur.As used herein, the heteroaryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, wherein at least one of the ringsin the ring system is fully unsaturated, i.e., it contains a cyclic,delocalized (4n+2) π-electron system in accordance with the Hückeltheory. Heteroaryl includes fused or bridged ring systems. Theheteroatom(s) in the heteroaryl radical is optionally oxidized. One ormore nitrogen atoms, if present, are optionally quaternized. Theheteroaryl is attached to the rest of the molecule through any atom ofthe ring(s). Examples of heteroaryls include, but are not limited to,azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl,benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl,benzo[b] [1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl,benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl,isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl,pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl,pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl,quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e.thienyl). Unless stated otherwise specifically in the specification, theterm “heteroaryl” is meant to include heteroaryl radicals as definedabove which are optionally substituted by one or more substituentsselected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl,haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl,optionally substituted aralkyl, optionally substituted aralkenyl,optionally substituted aralkynyl, optionally substituted carbocyclyl,optionally substituted carbocyclylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—R^(b)—OR^(a), —R^(b)-SR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—N(R^(a))₂,—R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)- C(O)N(R^(a))₂,—R^(b)—O—W—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a),—R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a)(where t is 1 or 2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2),where each R^(a) is independently hydrogen, alkyl, fluoroalkyl,cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl or heteroarylalkyl, each R^(b) isindependently a direct bond or a straight or branched alkylene oralkenylene chain, and R^(c) is a straight or branched alkylene oralkenylene chain, and where each of the above substituents isunsubstituted unless otherwise indicated.

“N-heteroaryl” refers to a heteroaryl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. An N-heteroaryl radical is optionallysubstituted as described above for heteroaryl radicals.

“C-heteroaryl” refers to a heteroaryl radical as defined above and wherethe point of attachment of the heteroaryl radical to the rest of themolecule is through a carbon atom in the heteroaryl radical. AC-heteroaryl radical is optionally substituted as described above forheteroaryl radicals.

“Heteroarylalkyl” refers to a radical of the formula —R^(c)-heteroaryl,where R^(c) is an alkylene chain as defined above. If the heteroaryl isa nitrogen-containing heteroaryl, the heteroaryl is optionally attachedto the alkyl radical at the nitrogen atom. The alkylene chain of theheteroarylalkyl radical is optionally substituted as defined above foran alkylene chain. The heteroaryl part of the heteroarylalkyl radical isoptionally substituted as defined above for a heteroaryl group.

The compounds, or their pharmaceutically acceptable salts may containone or more asymmetric centers and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)-for amino acids. When the compounds described herein contain olefinicdouble bonds or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both E(or trans) and Z (cis) geometric isomers. Likewise, all possibleisomers, as well as their racemic and optically pure forms, and alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. It is therefore contemplated that variousstereoisomers and mixtures thereof and includes “enantiomers,” whichrefers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The compounds presented herein mayexist as tautomers. Tautomers are compounds that are interconvertible bymigration of a hydrogen atom, accompanied by a switch of a single bondand adjacent double bond. In solutions where tautomerization ispossible, a chemical equilibrium of the tautomers will exist. The exactratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Some examples of tautomeric pairs include:

“Optional” or “optionally” means that a subsequently described event orcircumstance may or may not occur and that the description includesinstances when the event or circumstance occurs and instances in whichit does not.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts. A pharmaceutically acceptable salt of any one of thealkoxyphenyl-linked amine derivative compounds described herein isintended to encompass any and all pharmaceutically suitable salt forms.Preferred pharmaceutically acceptable salts of the compounds describedherein are pharmaceutically acceptable acid addition salts andpharmaceutically acceptable base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid,hydrofluoric acid, phosphorous acid, and the like. Also included aresalts that are formed with organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, alkanedioic acids, aromatic acids, aliphatic and. aromaticsulfonic acids, etc. and include, for example, acetic acid,trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Exemplary salts thus include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates,trifluoroacetates, propionates, caprylates, isobutyrates, oxalates,malonates, succinate suberates, sebacates, fumarates, maleates,mandelates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates,phenylacetates, citrates, lactates, malates, tartrates,methanesulfonates, and the like. Also contemplated are salts of aminoacids, such as arginates, gluconates, and galacturonates (see, forexample, Berge S. M. et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Science, 66:1-19 (1997), which is hereby incorporated byreference in its entirety). Acid addition salts of basic compounds maybe prepared by contacting the free base forms with a sufficient amountof the desired acid to produce the salt according to methods andtechniques with which a skilled artisan is familiar.

“Pharmaceutically acceptable base addition salt” refers to those saltsthat retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Pharmaceutically acceptable base addition salts may beformed with metals or amines, such as alkali and alkaline earth metalsor organic amines. Salts derived from inorganic bases include, but arenot limited to, sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, for example, isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, ethanolamine,diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline,betaine, ethylenediamine, ethylenedianiline, N-methylglucamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like. See Bergeet al., supra.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refers to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. By“therapeutic benefit” is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound described herein. Thus, the term “prodrug” refers to aprecursor of a biologically active compound that is pharmaceuticallyacceptable. A prodrug may be inactive when administered to a subject,but is converted in vivo to an active compound, for example, byhydrolysis. The prodrug compound often offers advantages of solubility,tissue compatibility or delayed release in a mammalian organism (see,e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam).

A discussion of prodrugs is provided in Higuchi, T_(max), et al.,“Pro-drugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14,and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche,American Pharmaceutical Association and Pergamon Press, 1987, both ofwhich are incorporated in full by reference herein.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound in vivo when such prodrug isadministered to a mammalian subject. Prodrugs of an active compound, asdescribed herein, may be prepared by modifying functional groups presentin the active compound in such a way that the modifications are cleaved,either in routine manipulation or in vivo, to the parent activecompound. Prodrugs include compounds wherein a hydroxy, amino ormercapto group is bonded to any group that, when the prodrug of theactive compound is administered to a mammalian subject, cleaves to forma free hydroxy, free amino or free mercapto group, respectively.Examples of prodrugs include, but are not limited to, acetate, formateand benzoate derivatives of alcohol or amine functional groups in theactive compounds and the like.

Peptides

The cystine-dense peptides herein can bind targets with antibody-likeaffinity. The cystine-dense peptides can modulate the activity of aplurality of cartilage regions, tissues, structures or cells. Forexample, in some embodiments, the cystine-dense peptide conjugated to abone-modifying drug homes to the cartilage of a diseased joint andreleases the drug, creating a higher local concentration of drug in anarea of eroded or damaged bone than would be achieved without thecartilage targeting function of the peptide. The cystine-dense peptidecan be conjugated to a drug that can affect nearby tissues or cells suchas the ligaments, muscle, tendons, bursa, connective tissue, bloodvessels, peripheral nerves, osteoclasts, osteoblasts, fibroblasts,synoviocytes, monocytes/macrophages, lymphocytes, plasma cells,adipocytes, endothelial cells, neurons, ligaments, muscle, tendons, andbursa. The cystine-dense peptide conjugated to a drug can bind to, hometo, migrate to, accumulate in, be retained by, or be directed tocartilage and its components, including chondrocytes, extracellularmatrix, collagen of any type, hyaluranon, aggrecan (also known ascartilage-specific proteoglycan core protein (CSPCP)), proteoglycans,glycoasminoglycans, glycoproteins, decorin, biclycan, fibromodulin, orother components of the extracellular matrix and the joint, or to othernearby components such as those described herein in joints andcartilaginous tissues as listed above. Some of the cartilage regions,tissues, and structures that peptides and peptide-drug conjugates cantarget to treat a cartilage-associated disorder include: (a) elasticcartilage; (b) hyaline cartilage, such as articular cartilage andphyseal cartilage; (c) fibrocartilage; and (d) any cells or cell typesin (a) -(c) above. Some of the areas where the peptide and peptide-drugconjugates can target to treat a cartilage-associated disorder include:cartilage includes joints such as knees, hips, or digits, nasalcartilage, spinal cartilage, tracheal cartilage, and rib cartilage. Invarious aspects, cartilage components include aggrecan and type IIcollagen. Additionally, in some embodiments, cystine-dense peptides canpenetrate into cells. In other embodiments, cystine-dense peptides donot enter cells. In other embodiments, cystine-dense peptides exhibitmore rapid clearance and cellular uptake compared to other types ofmolecules.

The peptides of the present disclosure can comprise cysteine amino acidresidues. In some cases, the peptide has at least 4 cysteine amino acidresidues. In some cases, the peptide has at least 6 cysteine amino acidresidues. In other cases, the peptide has at least 8 cysteine amino acidresidues, at least 10 cysteine amino acid residues, at least 12 cysteineamino acid residues, at least 14 cysteine amino acid residues or atleast 16 cysteine amino acid residues.

A cystine-dense peptide can comprise disulfide bridges. A cystine-densecan be a peptide wherein 5% or more of the residues are cysteinesforming intramolecular disulfide bonds as cystines. A disulfide-linkedpeptide can be a drug scaffold. In some embodiments, the disulfidebridges form an inhibitor knot. A disulfide bridge can be formed betweencysteine residues, for example, between cysteines 1 and 4, 2 and 5, or,3 and 6. In some cases, one disulfide bridge passes through a loopformed by the other two disulfide bridges, for example, to form theinhibitor knot. In other cases, the disulfide bridges can be formedbetween any two cysteine residues.

The present disclosure further includes peptide scaffolds that, e.g.,can be used as a starting point for generating additional peptides thatcan target and home to cartilage. In some embodiments, these scaffoldscan be derived from a variety of cystine-dense peptides. In certainembodiments, cystine-dense peptides are assembled into a complextertiary structure that is characterized by a number of intramoleculardisulfide crosslinks, and optionally contain beta strands and othersecondary structures such as an alpha helix. For example, cystine-densepeptides include, in some embodiments, small disulfide-rich proteinscharacterized by a disulfide through disulfide knot. This knot can be,e.g., obtained when one disulfide bridge crosses the macrocycle formedby two other disulfides and the interconnecting backbone. In someembodiments, the cystine-dense peptides can include growth factorcysteine knots or inhibitor cysteine knots. Other possible peptidestructures can include peptide having two parallel helices linked by twodisulfide bridges without β-sheets (e.g., hefutoxin).

A cystine-dense peptide can comprise at least one amino acid residue inan L configuration. A cystine-dense peptide can comprise at least oneamino acid residue in a D configuration. In some embodiments, acystine-dense peptide is 15-40 amino acid residues long. In otherembodiments, a cystine-dense peptide is 11-57 amino acid residues long.In further embodiments, a cystine-dense peptide is at least 20 aminoacid residues long.

In some embodiments, the peptides are members of the pfam00451:toxin_2family. The pfam00451:toxin_2 structural class family can include apeptide of any one of SEQ ID NO: 978-SEQ ID NO: 1024. A cartilage homingpeptide of this disclosure can be a variant of any peptide members ofthe pfam00451:toxin_2 family. In some embodiments, an exemplarycartilage homing peptide of this disclosure that is a variant of thepfam00451:toxin_2 structural class family is a peptide of SEQ ID NO:511. In other embodiments, an exemplary cartilage homing peptide of thisdisclosure that is a variant of the pfam00451:toxin_2 structural classfamily is a peptide of SEQ ID NO: 592. In other embodiments, the variantpeptides are at least 30% identical to a peptide of the structural classpfam00451:toxin_2 family. In some embodiments, the variant peptides are30%, 40%, 50%, 60%, 80%, 90% or 95% identical to a peptide of thestructural class pfam00451:toxin_2 family. In some embodiments, thevariant peptides are at least 30%, at least 40%, at least 50%, at least60%, at least 80%, at least 90% or at least 95% identical to a peptideof the structural class pfam00451:toxin_2 family.

In some embodiments, cartilage homing peptides are family members thesequences GSXVXXXVKCXGSKQCXXPCKRXXGXRXGKCINKKXCKCYXXX (SEQ ID NO: 493)or XVXXXVKCXGSKQCXXPCKRXXGXRXGKCINKKXCKCYXXX (SEQ ID NO: 767) wherein Xcan be any amino acid or amino acid analogue or null, in which thesesequences are based on the most common elements found in the followingsequences: GSGVPINVKCRGSRDCLDPCKKA-GMRFGKCINSK-CHCTP-(SEQ ID NO: 511),GS-VRIPVSCKHSGQCLKPCKDA-GMRFGKCMNGK-CDCTPK-(SEQ ID NO: 510),GSQVQTNVKCQGGS-CASVCRREIGVAAGKCINGK-CVCYRN-(SEQ ID NO: 514),GS-ISCTGSKQCYDPCKRKTGCPNAKCMNKS-CKCYGCG (SEQ ID NO: 513),GSEV-IRCSGSKQCYGPCKQQTGCTNSKCMNKV-CKCYGCG (SEQ ID NO: 515),GSAVCVYRT-CDKDCKRR-GYRSGKCINNA-CKCYPYG (SEQ ID NO: 512),GS-GIVC-KVCKIICGMQ-GKKVNICKAPIKCKCKKG-(SEQ ID NO: 508), andGSQIYTSKECNGSSECYSHCEGITGKRSGKCINKK-CYCYR-(SEQ ID NO: 517), where thefollowing residues may be independently interchanged in the sequences: Kand R; M, I, L, and V; G and A; S and T; Q and N; and X canindependently be any number of any amino acid or no amino acid. TheN-terminal GS sequence can be included or excluded between the peptidesof the present disclosure.

In some embodiments, cartilage homing peptides are family members of thesequences GSXVXIXVKCXGSXQCLXPCKXAXGXRXGKCMNGKCXCXPXX (SEQ ID NO: 505) orXVXIXVKCXGSXQCLXPCKXAXGXRXGKCMNGKCXCXPXX (SEQ ID NO: 779), wherein X canbe any amino acid or amino acid analogue or null, in which thesesequences are based on the most common elements found in the followingsequences:

-   VRIPVSCKHSGQCLKPCKDA-GMRFGKCMNGKCDCTPK-(SEQ ID NO: 800),    GVPINVKCRGSRDCLDPCKKA-GMRFGKCINSKCHCTP-(SEQ ID NO: 801),-   EVIRCSGSKQCYGPCKQQTGCTNSKCMNKVCKCYGCG (SEQ ID NO: 805),    GVIINVKCKISRQCLEPCKKA-GMRFGKCMNGKCHCTPK-(SEQ ID NO: 817),    GVPTDVKCRGSPQCIQPCKDA-GMRFGKCMNGKCHCTPK-(SEQ ID NO: 821),    GVPINVSCTGSPQCIKPCKDA-GMRFGKCMNRKCHCTPK- (SEQ ID NO: 822),-   VGINVKCKHSGQCLKPCKDA-GMRFGKCINGKCDCTPK-(SEQ ID NO: 824),    GVPINVRCRGSRDCLDPCRRA-GMRFGRCINSRCHCTP-(SEQ ID NO: 882),    QKILSNRCNNSSECIPHCIRIFGTRAAKCINRKCYCYP-(SEQ ID NO: 958),-   VFINVKCRGSPECLPKCKEAIGKSAGKCMNGKCKCYP-(SEQ ID NO: 967),-   VPTDVKCRGSPQCIQPCKDA-GMRFGKCMNGKCHCTP-(SEQ ID NO: 970),-   AEIIRCSGTRECYAPCQKLTGCLNAKCMNKACKCYGCV (SEQ ID NO: 1027),-   RPTDIKCSASYQCFPVCKSRFGKTNGRCVNGLCDCF-(SEQ ID NO: 1028),-   QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS-(SEQ ID NO: 1029),-   VGINVKCKHSRQCLKPCKDA-GMRFGKCTNGKCHCTPK-(SEQ ID NO: 1030),-   VVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDC-(SEQ ID NO: 1031),-   NFKVEGACSKPCRKYCIDK-GARNGKCINGRCHCYY-(SEQ ID NO: 1032), and    QIDTNVKCSGSSKCVKICIDRYNTRGAKCINGRCTCYP-(SEQ ID NO: 1033).

In some embodiments, the cartilage homing peptides are family members ofthe sequences GSXVXIXVRCXGSXQCLXPCRXAXGXRXGRCMNGRCXCXPXX (SEQ ID NO:506) or XVXIXVRCXGSXQCLXPCRXAXGXRXGRCMNGRCXCXPXX (SEQ ID NO: 780)wherein X can be any amino acid or amino acid analogue or null, in whichthese sequences are based on the most common elements found in thefollowing sequences and with K interchanged with R: SEQ ID NO: 800, SEQID NO: 801, SEQ ID NO: 805, SEQ ID NO: 817, SEQ ID NO: 821,SEQ ID NO:822, SEQ ID NO: 824, SEQ ID NO: 882, SEQ ID NO: 967, SEQ ID NO: 970, orSEQ ID NO: 1027—SEQ ID NO: 1033.

In some embodiments, a peptide comprises the sequenceGSGVPIX¹VRCRGSRDCX²X³PCRRAGX⁴RFGRCIX⁵X⁶RCX⁷CX⁸P (SEQ ID NO: 507) orGVPIX¹VRCRGSRDCX²X³PCRRAGX⁴RFGRCIX⁵X⁶RCX⁷CX⁸P (SEQ ID NO: 781), wherethe following residues where X¹ is selected from N, S, or G, wherein X²is selected from L or Y, wherein X³ is selected from D or E, wherein X⁴is selected from M or T, wherein X⁵ is selected from N, Q, A, S, T, orL, wherein X⁶ is selected from S, G, or R, wherein X⁷ is selected from Hor Y, and wherein X⁸ is selected from T or Y. In some embodiments, zeroor one or more of the R residues in SEQ ID NO: 507 or SEQ ID NO: 781 canbe replaced with K residues. In some embodiments, zero or one or more ofthe R residues in SEQ ID NO: 507 or SEQ ID NO: 781 can be replaced withA residues. In other embodiments, zero or one or more R residues in SEQID NO: 507 or SEQ ID NO: 781 can each be replaced with either a K or anA residue in any combination. In other embodiments, peptides are familymembers of the sequence GSXXXGCVXXXXKCRPGXKXCCXPXKRCSRRFGXXXXKKCKXXXXXX(SEQ ID NO: 494) or XXXGCVXXXXKCRPGXKXCCXPXKRCSRRFGXXXXKKCKXXXXXX (SEQID NO: 768), in which the sequence is based on the most common elementsfound in the following sequences:

-   GS-ACKGVFDACTPGKNECC-PNRVCSDK-H-KWCKWKL-(SEQ ID NO: 516),-   GS-GCLEFWWKCNPNDDKCCRPKLKCSKLF-KLCNFSFG-(SEQ ID NO: 518),-   GSSEKDCIKHLQRCR-ENKDCC-SKKCSRR-GTNPEKRCR-(SEQ ID NO: 509), and-   GS-GCFGY-KCDYY-KGCCSGYV-CSPTW-KWCVRPGPGR (SEQ ID NO: 520), where the    following residues may be independently interchanged in the    sequences: K and R; M, I, L, and V; G and A; S and T; Q and N; and X    can independently be any number of any amino acid or no amino acid.    The N-terminal GS sequence can be included or excluded between the    peptides of the present disclosure.

In some embodiments, a peptide comprises the sequenceGSGVX¹IX²X³KCX⁴GSKQCX⁵DPCKX⁶X⁷X⁸GX⁹RX¹⁰GKCX¹¹NKKCKCX¹²X¹³X¹⁴X¹⁵ (SEQ IDNO: 485) or

-   GVX¹IX²X³KCX⁴GSKQCX⁵DPCKX⁶X⁷X⁸GX⁹RX¹⁰GKCX¹¹NKKCKCX¹²X¹³X¹⁴X¹⁵(SEQ ID    NO: 759), wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹²,    X¹³, X¹⁴ and X¹⁵ are each individually any amino acid or amino acid    analogue or null. In some cases, the peptide comprises the sequence-   GSGVX¹IX²X³KCX⁴GSKQCX⁵DPCKX⁶X⁷X⁸GX⁹RX¹⁰GKCX¹¹NKKCKCX¹²X¹³X¹⁴X¹⁵ (SEQ    ID NO: 486) or-   GVX¹IX²X³KCX⁴GSKQCX⁵DPCKX⁶X⁷X⁸GX⁹RX¹⁰GKCX¹¹NKKCKCX¹²X¹²X¹³X¹⁴X¹⁵    (SEQ ID NO: 760), where X¹ is selected from P or R, wherein X² is    selected from P or N, wherein X³ is selected from V or I, wherein X⁴    is selected from S, T, R or K, wherein X⁵ is selected from Y or L,    wherein X⁶ is selected from Q, R or K, wherein X⁷ is selected from    A, K or R, wherein X⁸ is selected from T or A, wherein X⁹ is    selected from C or M, wherein X¹⁰ is selected from F or N, wherein    X¹¹ is selected from M or I, wherein X¹² is selected from Y or T,    wherein X¹³ is selected from G or P, wherein X¹⁴ is selected from C    or null, and wherein X¹⁵ is selected from G or null.

In some embodiments, a peptide comprises the sequence

-   GSX¹X²X³X⁴IX⁵CX⁶GSKQCYX⁷PCKX⁸X⁹TGCX¹⁰X¹¹X¹²KCX¹³X¹⁴KX¹⁵CKCYGCG (SEQ    ID NO: 487) or-   X¹X²X³X⁴IX⁵CX⁶GSKQCYX⁷PCKX⁸X⁹TGCX¹⁰X¹¹X¹²KCX¹³X¹⁴CKCYGCG (SEQ ID NO:    761), wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, _(X) ⁹ _(, X) ¹⁰, X¹¹,    X¹², X¹³, X¹⁴, and X¹⁵ are each individually any amino acid or amino    acid analogue or null. In some cases, the peptide comprises the    sequence-   GSX¹X²X³X⁴IX⁵CX⁶GSKQCYX⁷PCKX⁸X⁹TGCX¹⁰X¹¹X¹²KCX¹³X¹⁴KX¹⁵CKCYGCG (SEQ    ID NO: 488) or-   X¹X²X³X⁴IX⁵CX⁶GSKQCYX⁷PCKX⁸X⁹TGCX¹⁰X¹¹X¹²KCX¹³X¹⁴KX¹⁵CKCYGCG (SEQ ID    NO: 762), where X¹ is selected from G or null, wherein X² is    selected from S or null, wherein X³ is selected from E, G or null,    wherein X⁴ is selected from V, S, or null, wherein X⁵ is selected    from R or S, wherein X⁶ is selected from S or T, wherein X⁷ is    selected from G or D, wherein X⁸ is selected from Q or R, wherein X⁹    is selected from Q or K, wherein X¹⁰ is selected from T or P,    wherein X¹¹ is selected from N or Q, wherein X¹² is selected from S    or A, wherein X¹³ is selected from M or L, wherein X¹⁴ is selected    from N or Q, and wherein X¹⁵ is selected from V or S.

In some embodiments, a peptide comprises the sequence

-   GSX¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCKX¹²AGMRFGKCX¹³NX¹⁴KCX¹⁵CTPX¹⁶    (SEQ ID NO: 489) or-   X¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCKX¹²AGMRFGKCX¹³NX¹⁴KCX¹⁵CTPX¹⁶ (SEQ    ID NO: 763), wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, _(X) ⁹, X¹⁰,    X¹¹, X¹², X¹³, X¹⁴, X¹⁵, X¹⁶ are each individually any amino acid or    amino acid analogue or null. In some cases, the peptide comprises    the sequence-   GSX¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCKX¹²AGMRFGKCX¹³NX¹⁴KCX¹⁵CTPX¹⁶    (SEQ ID NO: 490) or-   X¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCKX¹²AGMRFGKCX¹³NX¹⁴KCX¹⁵CTPX¹⁶ (SEQ    ID NO: 764), where X¹ is selected from G or null, wherein X² is    selected from G, S or null, wherein X³ is selected from G, S or    null, wherein X⁴ is selected from P or R, wherein X⁵ is selected    from N or P, wherein X⁶ is selected from K or S, wherein X⁷ is    selected from R or K, wherein X⁸ is selected from G or H, wherein X⁹    is selected from R or G, wherein X¹⁰ is selected from D or Q,    wherein X¹¹ is selected from D or K, wherein X¹² is selected from K    or D, wherein X¹³ is selected from I or M, wherein X¹⁴ is selected    from S or G, wherein X¹⁵ is selected from H or D, and wherein X¹⁶ is    selected from K or null.

In some embodiments, a peptide comprises the sequence

-   GSXVXVKCXGSKQCXPCKRXGXRXGKCINKKXCKCYX (SEQ ID NO: 491) or-   GSXGCVXKCRPGXKXCCXPXKRCSRRFGXKKCKX (SEQ ID NO: 492), wherein each    letter is each individually any amino acid or amino acid analogue    and where X is no amino acid or a 1-10 amino acid long peptide    fragment wherein each amino acid within such peptide fragment can in    each case be any amino acid or amino acid analogue. In some    embodiments, a peptide comprises the sequence    XVXVKCXGSKQCXPCKRXGXRXGKCINKKXCKCYX (SEQ ID NO: 765) or    XGCVXKCRPGXKXCCXPXKRCSRRFGXKKCKX (SEQ ID NO: 766), wherein each    letter is each individually any amino acid or amino acid analogue    and where X is no amino acid or a 1-10 amino acid long peptide    fragment wherein each amino acid within such peptide fragment can in    each case be any amino acid or amino acid analogue.

In some embodiments, a peptide comprises the sequence

-   GSGVX¹IX²X³RCX⁴GSRQCX⁵DPCRX⁶X⁷X⁸GX⁹RX¹⁰GRCX¹¹NRRCRCX¹²X¹³X¹⁴X¹⁵ (SEQ    ID NO: 495) or-   GVX¹IX²X³RCX⁴GSRQCX⁵DPCRX⁶X⁷X⁸GX⁹RX¹⁰GRCX¹¹NRRCRCX¹²X¹³X¹⁴, and    X¹⁵(SEQ ID NO: 769), wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹,    X¹⁰, X¹¹, X¹², X¹³, X¹⁴, and X¹⁵ are each individually any amino    acid or amino acid analogue or null. In some cases, the peptide    comprises the sequence-   GSGVX¹IX²X³RCX⁴GSRQCX⁵DPCRX⁶X⁷X⁸GX⁹RX¹¹⁰GRCX¹¹NRRCRCX¹²X¹³X¹⁴X¹⁵    (^(SEQ) ID NO: 496) or-   GVX¹IX²X³RCX⁴GSRQCX⁵DPCRX⁶X⁷X⁸GX⁹RX¹⁰GRCX¹¹NRRCRCX¹²X¹³X¹⁴X¹⁵ (SEQ    ID NO: 770), where X¹ is selected from P or R, wherein X² is    selected from P or N, wherein X³ is selected from V or I, wherein X⁴    is selected from S, T, R or K, wherein X⁵ is selected from Y or L,    wherein X⁶ is selected from Q, R or K, wherein X⁷ is selected from    A, K or R, wherein X⁸ is selected from T or A, wherein X⁹ is    selected from C or M, wherein X¹⁰ is selected from F or N, wherein    X¹¹ is selected from M or I, wherein X¹² is selected from Y or T,    wherein X¹³ is selected from G or P, wherein X¹⁴ is selected from C    or null, and wherein X¹⁵ is selected from G or null.

In some embodiments, a peptide comprises the sequence

-   GSX¹X²X³X⁴IX⁵CX⁶GSRQCYX⁷PCRX⁸X⁹TGCX¹⁰X¹¹X¹²RCX¹³X¹⁴RX¹⁵ CRCYGCG (SEQ    ID NO: 497) or    X¹X²X³X⁴IX⁵CX⁶GSRQCYX⁷PCRX⁸X⁹TGCX¹⁰X¹¹X¹²RCX¹³X¹⁴RX¹⁵CRCYGCG (SEQ ID    NO: 771), wherein X^(l), X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹,    X¹², X¹³, X¹⁴, and X¹⁵are each individually any amino acid or amino    acid analogue or null. In some cases, the peptide comprises the    sequence-   GSX¹X²X³X⁴IX⁵CX⁶GSRQCYX⁷PCRX⁸X⁹TGCX¹⁰X¹¹X¹²RCX¹³X¹⁴RX¹⁵CRCYGCG, (SEQ    ID NO: 498) or    X¹X²X³X⁴IX⁵CX⁶GSRQCYX⁷PCRX⁸X⁹TGCX¹⁰X¹¹X¹²RCX¹³X¹⁴X¹⁴RX¹⁵ CRCYGCG    (SEQ ID NO: 772), where X¹ is selected from G or null, wherein X² is    selected from S or null, wherein X³ is selected from E, G or null,    wherein X⁴ is selected from V, S, or null, wherein X⁵ is selected    from R or S, wherein X⁶ is selected from S or T, wherein X⁷ is    selected from G or D, wherein X⁸ is selected from Q or R, wherein X⁹    is selected from Q, R, or K, wherein X¹⁰ is selected from T or P,    wherein X¹¹ is selected from N or Q, wherein X¹² is selected from S    or A, wherein X¹³ is selected from M or L, wherein X¹⁴ is selected    from N or Q, and wherein X¹⁵ is selected from V or S.

In some embodiments, a peptide comprises the sequence

-   GSX¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCRX¹²AGMRFGRCX¹³NX¹⁴RCX¹⁵CTPX (SEQ    ID NO: 499) or-   X¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCRX¹²AGMRFGRCX¹³NX¹⁴RCX¹⁵CTPX (SEQ    ID NO: 773), wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹,    X¹², X¹³, X¹⁴, X¹⁵, X¹⁶ are each individually any amino acid or    amino acid analogue or null. In some cases, the peptide comprises    the sequence-   GSX¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCRX¹²AGMRFGRCX¹³NX¹⁴RCX¹⁵CTPX¹⁶    (SEQ ID NO: 500) or-   X¹X²X³VX⁴IX⁵VX⁶CX⁷X⁸SX⁹X¹⁰CLX¹¹PCRX¹²AGMRFGRCX¹³NX¹⁴RCX¹⁵CTPX¹⁶ (SEQ    ID NO: 774), where X¹ is selected from G or null, wherein X² is    selected from G, S or null, wherein X³ is selected from G, S or    null, wherein X⁴ is selected from P or R, wherein X⁵ is selected    from N or P, wherein X⁶ is selected from R, K or S, wherein X⁷ is    selected from R or K, wherein X⁸ is selected from G or H, wherein X⁹    is selected from R or G, wherein X¹⁰ is selected from D or Q,    wherein X¹¹ is selected from D, R, or K, wherein X¹² is selected    from K, R, or D, wherein X¹³ is selected from I or M, wherein X¹⁴ is    selected from S or G, wherein X¹⁵ is selected from H or D, and    wherein X¹⁶ is selected from K, R, or null.

In some embodiments, a peptide comprises the sequence

-   GSXVXVRCXGSRQCXPCRRXGXRXGRCINRRXCRCYX (SEQ ID NO: 501) or-   GSXGCVXRCRPGXRXCCXPXRRCSRRFGXRRCRX (SEQ ID NO: 502), wherein each    letter is each individually any amino acid or amino acid analogue    and where X is no amino acid or a 1-10 amino acid long peptide    fragment wherein each amino acid within such peptide fragment can in    each case be any amino acid or amino acid analogue. In some    embodiments, a peptide comprises the sequence    XVXVRCXGSRQCXPCRRXGXRXGRCINRRXCRCYX (SEQ ID NO: 775) or    XGCVXRCRPGXRXCCXPXRRCSRRFGXRRCRX (SEQ ID NO: 776), wherein each    letter is each individually any amino acid or amino acid analogue    and where X is no amino acid or a 1-10 amino acid long peptide    fragment wherein each amino acid within such peptide fragment can in    each case be any amino acid or amino acid analogue.

In some embodiments, a peptide comprises the sequence

-   GSXVXXXVRCXGSRQCXXPCRRXXGXRXGRCINRRXCRCYXXX (SEQ ID NO: 503),-   XVXXXVRCXGSRQCXXPCRRXXGXRXGRCINRRXCRCYXXX (SEQ ID NO: 777),-   GSXXXGCVXXXXRCRPGXRXCCXPXRRCSRRFGXXXXRRCRXXXXXX (SEQ ID NO: 504), or    XXXGCVXXXXRCRPGXRXCCXPXRRCSRRFGXXXXRRCRXXXXXX (SEQ ID NO: 778)    wherein X is no amino acid or any amino acid analogue.

In some embodiments, a peptide comprises the one or more of thefollowing peptide fragments: GKCMNGKC (SEQ ID NO: 796); GRCMNGRC (SEQ IDNO: 797); GKCINKKCKC (SEQ ID NO: 782); KCIN (SEQ ID NO: 783); KKCK (SEQID NO: 784); PCKR (SEQ ID NO: 785); KRCSRR (SEQ ID NO: 786); KQC (SEQ IDNO: 787); GRCINRRCRC (SEQ ID NO: 788); RCIN (SEQ ID NO: 789); RRCR (SEQID NO: 790); PCRR (SEQ ID NO: 791); RRCSRR (SEQ ID NO: 792); RQC (SEQ IDNO: 793); PCKK (SEQ ID NO: 794), and KKCSKK (SEQ ID NO: 795).

TABLE 51 lists some exemplary peptides according to the presentdisclosure.

TABLE 51  Exemplary Amino Acid Sequences SEQ ID NO: Amino Acid SequenceSEQ ID NO: 508 GSGIVCKVCKIICGMQGKKVNICKAPIKCKCKKG SEQ ID NO: 509GSSEKDCIKHLQRCRENKDCCSKKCSRRGTNPEKRCR SEQ ID NO: 510GSVRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK SEQ ID NO: 511GSGVPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP SEQ ID NO: 512GSAVCVYRTCDKDCKRRGYRSGKCINNACKCYPYG SEQ ID NO: 513GSISCTGSKQCYDPCKRKTGCPNAKCMNKSCKCYGCG SEQ ID NO: 514GSQVQTNVKCQGGSCASVCRREIGVAAGKCINGKCVCYRN SEQ ID NO: 515GSEVIRCSGSKQCYGPCKQQTGCTNSKCMNKVCKCYGCG SEQ ID NO: 516GSACKGVFDACTPGKNECCPNRVCSDKHKWCKWKL SEQ ID NO: 517GSQIYTSKECNGSSECYSHCEGITGKRSGKCINKKCYCYR SEQ ID NO: 518GSGCLEFWWKCNPNDDKCCRPKLKCSKLFKLCNFSFG SEQ ID NO: 519GSDCVRFWGKCSQTSDCCPHLACKSKWPRNICVWDGSVG SEQ ID NO: 520GSGCFGYKCDYYKGCCSGYVCSPTWKWCVRPGPGR SEQ ID NO: 521GSMNAKFILLLVLTTMMLLPDTKGAEVIRCSGSKQCYGPCKQQTGCTNSK CMNKVCKCYGCGSEQ ID NO: 522 GSMNAKLIYLLLVVTTMTLMFDTAQAVDIMCSGPKQCYGPCKKETGCPNAKCMNRRCKCYGCV SEQ ID NO: 523GSMNAKLIYLLLVVTTMMLTFDTTQAGDIKCSGTRQCWGPCKKQTTCTNS KCMNGKCKCYGCVGSEQ ID NO: 524 GSMNTKFIFLLLVVTNTMMLFDTKPVEGISCTGSKQCYDPCKRKTGCPNAKCMNKSCKCYGCG SEQ ID NO: 525 GSGVPINVKCSGSRDCLEPCKKAGMRFGKCINRKCHCTPKSEQ ID NO: 526 GSGVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHCTPK SEQ ID NO: 527GSGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK SEQ ID NO: 528GSGVPINVKCRGSPQCIQPCRDAGMRFGKCMNGKCHCTPQ SEQ ID NO: 529GSGVEINVKCTGSHQCIKPCKDAGMRFGKCINRKCHCTPK SEQ ID NO: 530GSGVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK SEQ ID NO: 531GSGVPTDVKCRGSPQCIQPCKDAGMRFGKCMNGKCHCTPK SEQ ID NO: 532GSGVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK SEQ ID NO: 533GSGVPINVPCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK SEQ ID NO: 534GSVGINVKCKHSGQCLKPCKDAGMRFGKCINGKCDCTPK SEQ ID NO: 535GSVGINVKCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK SEQ ID NO: 536GSVGIPVSCKHSGQCIKPCKDAGMRFGKCMNRKCDCTPK SEQ ID NO: 537GSRKGCFKEGHSCPKTAPCCRPLVCKGPSPNTKKCTRP SEQ ID NO: 538GSSFCIPFKPCKSDENCCKKFKCKTTGIVKLCRW SEQ ID NO: 539GSLKGCLPRNRFCNALSGPRCCSGLRCKELSIWASKCL SEQ ID NO: 540GSGNYCLRGRCLPGGRKCCNGRPCECFAKICSCKPK SEQ ID NO: 541GSTVKCGGCNRKCCPGGCRSGKCINGKCQCY SEQ ID NO: 542GSGCMKEYCAGQCRGKVSQDYCLKHCKCIPR SEQ ID NO: 543GSACLGFGEKCNPSNDKCCKSSSLVCSQKHKWCKYG SEQ ID NO: 544GSRGGCLPHNRFCNALSGPRCCSGLRCKELSIRDSRCLG SEQ ID NO: 545GSRGGCLPRNKFCNPSSGPRCCSGLTCKELNIWASKCL SEQ ID NO: 546GSQRSCAKPGDMCMGIKCCDGQCGCNRGTGRCFCK SEQ ID NO: 547GSARGCADAYKSCNHPRTCCDGYNGYKRACICSGSNCKCKKS SEQ ID NO: 548GSRGGCLPHNRFCNALSGPRCCSGLRCKELSIWDSRCLG SEQ ID NO: 549GSRGGCLPHNRFCNALSGPRCCSGLKCKELSIYDSRCLG SEQ ID NO: 550GSRGGCLPHNRFCNALSGPRCCSRLKCKELSIWDSRCLG SEQ ID NO: 551GSRGGCLPHNRFCNALTGPRCCSRLRCKELSIWDSICLG SEQ ID NO: 552GSSCADAYKSCDSLKCCNNRTCMCSMIGTNCTCRKK SEQ ID NO: 553GSERRCLPAGKTCVRGPMRVPCCGSCSQNKCT SEQ ID NO: 554GSLCSREGEFCYKLRKCCAGFYCKAFVLHCYRN SEQ ID NO: 555GSACGSCRKKCKGSGKCINGRCKCY SEQ ID NO: 556 GSACGSCRKKCKGPGKCINGRCKCYSEQ ID NO: 557 GSACQGYMRKCGRDKPPCCKKLECSKTWRWCVWN SEQ ID NO: 558GSGRYCQKWMWTCDSKRACCEGLRCKLWCRKI SEQ ID NO: 559GSNAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP SEQ ID NO: 560GSNVKCRGSKECLPACKAAVGKAAGKCMNGKCKCYP SEQ ID NO: 561GSNVKCRGSPECLPKCKEAIGKSAGKCMNGKCKCYP SEQ ID NO: 562GSNAKCRGSPECLPKCKQAIGKAAGKCMNGKCKCYP SEQ ID NO: 563GSRGYCAEKGIKCHNIHCCSGLTCKCKGSSCVCRK SEQ ID NO: 564GSERGCKLTFWKCKNKKECCGWNACALGICMPR SEQ ID NO: 565GSKKKCIAKDYGRCKWGGTPCCRGRGCICSIMGTNCECKPR SEQ ID NO: 566GSGCKLTFWKCKNKKECCGWNACALGICMPR SEQ ID NO: 567GSACKGLFVTCTPGKDECCPNHVCSSKHKWCKYK SEQ ID NO: 568GSIACAPRGLLCFRDKECCKGLTCKGRFVNTWPTFCLV SEQ ID NO: 569GSACAGLYKKCGKGVNTCCENRPCKCDLAMGNCICKKK SEQ ID NO: 570GSFTCAISCDIKVNGKPCKGSGEKKCSGGWSCKFNVCVKV SEQ ID NO: 571GSGFCAQKGIKCHDIHCCTNLKCVREGSNRVCRKA SEQ ID NO: 572GSCAKKRNWCGKNEDCCCPMKCIYAWYNQQGSCQSTITGLFKKC SEQ ID NO: 573GSYCQKWMWTCDSARKCCEGLVCRLWCKKI SEQ ID NO: 574GSRGGCLPHNKFCNALSGPRCCSGLKCKELTIWNTKCLE SEQ ID NO: 575GSNVKCTGSKQCLPACKAAVGKAAGKCMNGKCKCYT SEQ ID NO: 576GSQRSCAKPGEMCMRIKCCDGQCGCNRGTGRCFCK SEQ ID NO: 577GSGCIPKHKRCTWSGPKCCNNISCHCNISGTLCKCRPG SEQ ID NO: 578GSNYCVAKRCRPGGRQCCSGKPCACVGKVCKCPRD SEQ ID NO: 579GSERGCSGAYKRCSSSQRCCEGRPCVCSAINSNCKCRKT SEQ ID NO: 580GSRYCPRNPEACYNYCLRTGRPGGYCGGRSRITCFCFR SEQ ID NO: 581GSQRSCAKPGEMCMGIKCCDGQCGCNRGTGRCFCK SEQ ID NO: 582GSRRGCFKEGKWCPKSAPCCAPLKCKGPSIKQQKCVRE SEQ ID NO: 583GSTVKCGGCNRKCCAGGCRSGKCINGKCQCYGR SEQ ID NO: 584GSERRCEPSGKPCRPLMRIPCCGSCVRGKCA SEQ ID NO: 585GSRGGCLPRNKFCNPSSGPRCCSGLTCKELNIWANKCL SEQ ID NO: 586GSCAKKRNWCGKNEDCCCPMKCIYAWYNQQGSCQTTITGLFKKC SEQ ID NO: 587GSVRIPVSCKHSGQCLKPCKDAGMRTGKCMNGKCDCTPK SEQ ID NO: 588GSVKCTTSKDCWPPCKKVTGRA SEQ ID NO: 589 GSGIVCRVCRIICGMQGRRVNICRAPIRCRCRRGSEQ ID NO: 590 GSSERDCIRHLQRCRENRDCCSRRCSRRGTNPERRCR SEQ ID NO: 591GSVRIPVSCRHSGQCLRPCRDAGMRFGRCMNGRCDCTPR SEQ ID NO: 592GSGVPINVRCRGSRDCLDPCRRAGMRFGRCINSRCHCTP SEQ ID NO: 593GSAVCVYRTCDRDCRRRGYRSGRCINNACRCYPYG SEQ ID NO: 594GSISCTGSRQCYDPCRRRTGCPNARCMNRSCRCYGCG SEQ ID NO: 595GSQVQTNVRCQGGSCASVCRREIGVAAGRCINGRCVCYRN SEQ ID NO: 596GSEVIRCSGSRQCYGPCRQQTGCTNSRCMNRVCRCYGCG SEQ ID NO: 597GSACRGVFDACTPGRNECCPNRVCSDRHRWCRWRL SEQ ID NO: 598GSQIYTSRECNGSSECYSHCEGITGRRSGRCINRRCYCYR SEQ ID NO: 599GSGCLEFWWRCNPNDDRCCRPRLRCSRLFRLCNFSFG SEQ ID NO: 600GSDCVRFWGRCSQTSDCCPHLACRSRWPRNICVWDGSVG SEQ ID NO: 601GSGCFGYRCDYYRGCCSGYVCSPTWRWCVRPGPGR SEQ ID NO: 602GSMNARFILLLVLTTMMLLPDTRGAEVIRCSGSRQCYGPCRQQTGCTNSRC MNRVCRCYGCGSEQ ID NO: 603 GSMNARLIYLLLVVTTMTLMFDTAQAVDIMCSGPRQCYGPCRRETGCPNARCMNRRCRCYGCV SEQ ID NO: 604GSMNARLIYLLLVVTTMMLTFDTTQAGDIRCSGTRQCWGPCRRQTTCTNS RCMNGRCRCYGCVGSEQ ID NO: 605 GSMNTRFIFLLLVVTNTMMLFDTRPVEGISCTGSRQCYDPCRRRTGCPNARCMNRSCRCYGCG SEQ ID NO: 606 GSGVPINVRCSGSRDCLEPCRRAGMRFGRCINRRCHCTPRSEQ ID NO: 607 GSGVPINVRCTGSPQCLRPCRDAGMRFGRCINGRCHCTPR SEQ ID NO: 608GSGVIINVRCRISRQCLEPCRRAGMRFGRCMNGRCHCTPR SEQ ID NO: 609GSGVPINVRCRGSPQCIQPCRDAGMRFGRCMNGRCHCTPQ SEQ ID NO: 610GSGVEINVRCTGSHQCIRPCRDAGMRFGRCINRRCHCTPR SEQ ID NO: 611GSGVEINVRCSGSPQCLRPCRDAGMRFGRCMNRRCHCTPR SEQ ID NO: 612GSGVPTDVRCRGSPQCIQPCRDAGMRFGRCMNGRCHCTPR SEQ ID NO: 613GSGVPINVSCTGSPQCIRPCRDAGMRFGRCMNRRCHCTPR SEQ ID NO: 614GSGVPINVPCTGSPQCIRPCRDAGMRFGRCMNRRCHCTPR SEQ ID NO: 615GSVGINVRCRHSGQCLRPCRDAGMRFGRCINGRCDCTPR SEQ ID NO: 616GSVGINVRCRHSGQCLRPCRDAGMRFGRCMNGRCDCTPR SEQ ID NO: 617GSVGIPVSCRHSGQCIRPCRDAGMRFGRCMNRRCDCTPR SEQ ID NO: 618GSRRGCFREGHSCPRTAPCCRPLVCRGPSPNTRRCTRP SEQ ID NO: 619GSSFCIPFRPCRSDENCCRRFRCRTTGIVRLCRW SEQ ID NO: 620GSLRGCLPRNRFCNALSGPRCCSGLRCRELSIWASRCL SEQ ID NO: 621GSGNYCLRGRCLPGGRRCCNGRPCECFARICSCRPR SEQ ID NO: 622GSTVRCGGCNRRCCPGGCRSGRCINGRCQCY SEQ ID NO: 623GSGCMREYCAGQCRGRVSQDYCLRHCRCIPR SEQ ID NO: 624GSACLGFGERCNPSNDRCCRSSSLVCSQRHRWCRYG SEQ ID NO: 625GSRGGCLPHNRFCNALSGPRCCSGLRCRELSIRDSRCLG SEQ ID NO: 626GSRGGCLPRNRFCNPSSGPRCCSGLTCRELNIWASRCL SEQ ID NO: 627GSQRSCARPGDMCMGIRCCDGQCGCNRGTGRCFCR SEQ ID NO: 628GSARGCADAYRSCNHPRTCCDGYNGYRRACICSGSNCRCRRS SEQ ID NO: 629GSRGGCLPHNRFCNALSGPRCCSGLRCRELSIWDSRCLG SEQ ID NO: 630GSRGGCLPHNRFCNALSGPRCCSGLRCRELSIYDSRCLG SEQ ID NO: 631GSRGGCLPHNRFCNALSGPRCCSRLRCRELSIWDSRCLG SEQ ID NO: 632GSRGGCLPHNRFCNALTGPRCCSRLRCRELSIWDSICLG SEQ ID NO: 633GSSCADAYKSCDSLRCCNNRTCMCSMIGTNCTCRRR SEQ ID NO: 634GSERRCLPAGRTCVRGPMRVPCCGSCSQNRCT SEQ ID NO: 635GSLCSREGEFCYRLRRCCAGFYCRAFVLHCYRN SEQ ID NO: 636GSACGSCRRRCRGSGRCINGRCRCY SEQ ID NO: 637 GSACGSCRRRCRGPGRCINGRCRCYSEQ ID NO: 638 GSACQGYMRRCGRDRPPCCRRLECSRTWRWCVWN SEQ ID NO: 639GSGRYCQRWMWTCDSRRACCEGLRCRLWCRRI SEQ ID NO: 640GSNARCRGSPECLPRCREAIGRAAGRCMNGRCRCYP SEQ ID NO: 641GSNVRCRGSRECLPACRAAVGRAAGRCMNGRCRCYP SEQ ID NO: 642GSNVRCRGSPECLPRCREAIGRSAGRCMNGRCRCYP SEQ ID NO: 643GSNARCRGSPECLPRCRQAIGRAAGRCMNGRCRCYP SEQ ID NO: 644GSRGYCAERGIRCHNIHCCSGLTCRCRGSSCVCRR SEQ ID NO: 645GSERGCRLTFWRCRNRRECCGWNACALGICMPR SEQ ID NO: 646GSRRRCIARDYGRCRWGGTPCCRGRGCICSIMGTNCECRPR SEQ ID NO: 647GSGCRLTFWRCRNRRECCGWNACALGICMPR SEQ ID NO: 648GSACRGLFVTCTPGRDECCPNHVCSSRHRWCRYR SEQ ID NO: 649GSIACAPRGLLCFRDRECCRGLTCRGRFVNTWPTFCLV SEQ ID NO: 650GSACAGLYRRCGRGVNTCCENRPCRCDLAMGNCICRRR SEQ ID NO: 651GSFTCAISCDIRVNGRPCRGSGERRCSGGWSCRFNVCVRV SEQ ID NO: 652GSGFCAQRGIRCHDIHCCTNLRCVREGSNRVCRRA SEQ ID NO: 653GSCARRRNWCGRNEDCCCPMRCIYAWYNQQGSCQSTITGLFRRC SEQ ID NO: 654GSYCQRWMWTCDSARRCCEGLVCRLWCRRI SEQ ID NO: 655GSRGGCLPHNRFCNALSGPRCCSGLRCRELTIWNTRCLE SEQ ID NO: 656GSNVRCTGSRQCLPACRAAVGRAAGRCMNGRCRCYT SEQ ID NO: 657GSQRSCARPGEMCMRIRCCDGQCGCNRGTGRCFCR SEQ ID NO: 658GSGCIPRHRRCTWSGPRCCNNISCHCNISGTLCRCRPG SEQ ID NO: 659GSNYCVARRCRPGGRQCCSGRPCACVGRVCRCPRD SEQ ID NO: 660GSERGCSGAYRRCSSSQRCCEGRPCVCSAINSNCRCRRT SEQ ID NO: 661GSQRSCARPGEMCMGIRCCDGQCGCNRGTGRCFCR SEQ ID NO: 662GSRRGCFREGRWCPRSAPCCAPLRCRGPSIRQQRCVRE SEQ ID NO: 663GSTVRCGGCNRRCCAGGCRSGRCINGRCQCYGR SEQ ID NO: 664GSERRCEPSGRPCRPLMRIPCCGSCVRGRCA SEQ ID NO: 665GSRGGCLPRNRFCNPSSGPRCCSGLTCRELNIWANRCL SEQ ID NO: 666GSCARRRNWCGRNEDCCCPMRCIYAWYNQQGSCQTTITGLFRRC SEQ ID NO: 667GSVRIPVSCRHSGQCLRPCRDAGMRTGRCMNGRCDCTPR SEQ ID NO: 668GSQKILSNRCNNSSECIPHCIRIFGTRAAKCINRKCYCYP SEQ ID NO: 669GSAVCNLKRCQLSCRSLGLLGKCIGDKCECVKHG SEQ ID NO: 670GSISIGIRCSPSIDLCEGQCRIRRYFTGYCSGDTCHCSG SEQ ID NO: 671GSGDCLPHLRRCRENNDCCSRRCRRRGANPERRCR SEQ ID NO: 672GSSCEPGRTFRDRCNTCKCGADGRSAACTLRACPNQ SEQ ID NO: 673GSGDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR SEQ ID NO: 674GSGDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR SEQ ID NO: 675GSKDCLKKLKLCKENKDCCSKSCKRRGTNIEKRCR SEQ ID NO: 676GSGDCLPHLKRCKENNDCCSKKCKRRGANPEKRCR SEQ ID NO: 677GSVFINVKCRGSPECLPKCKEAIGKSAGKCMNGKCKCYP SEQ ID NO: 678GSVFINAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP SEQ ID NO: 679GSVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP SEQ ID NO: 680GSVPTDVKCRGSPQCIQPCKDAGMRFGKCMNGKCHCTP SEQ ID NO: 681GSVRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTP SEQ ID NO: 682GSVRIPVSCRHSGQCLRPCRDAGMRFGRCMNGRCDCTP SEQ ID NO: 683GSTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRC SEQ ID NO: 684GSNVKCTGSKQCLPACKAAVGKAAGKCMNGKCKC SEQ ID NO: 685GSGVPINVRCRGSRDCLDPCRGAGERHGRCGNSRCHCTP SEQ ID NO: 686GSVRIPVSCRHSGQCLRPCRDAGERHGRCGGGRCDCTPR SEQ ID NO: 687GSQVQTNVRCQGGSCGSVCRREGGGAGGGCGNGRCGCYRN SEQ ID NO: 688GSIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF SEQ ID NO: 689GSVKCSSPQQCLKPCKAAFGISAGGKCINGKCKCY SEQ ID NO: 690GSVSCSASSQCWPVCKKLFGTYRGKCMNSKCRCY SEQ ID NO: 691GSESCTASNQCWSICKRLHNTNRGKCMNKKCRCY SEQ ID NO: 692GSVSCTTSKECWSVCEKLYNTSRGKCMNKKCRCY SEQ ID NO: 693GSMRCKSSKECLVKCKQATGRPNGKCMNRKCKCY SEQ ID NO: 694GSIKCTLSKDCYSPCKKETGCPRAKCINRNCKCY SEQ ID NO: 695GSIRCSGSRDCYSPCMKQTGCPNAKCINKSCKCY SEQ ID NO: 696GSIRCSGTRECYAPCQKLTGCLNAKCMNKACKCY SEQ ID NO: 697GSISCTNPKQCYPHCKKETGYPNAKCMNRKCKCF SEQ ID NO: 698GSASCRTPKDCADPCRKETGCPYGKCMNRKCKCN SEQ ID NO: 699GSTSCISPKQCTEPCRAKGCKHGKCMNRKCHCM SEQ ID NO: 700GSKECTGPQHCTNFCRKN-KCTHGKCMNRKCKCF SEQ ID NO: 701GSIKCRTPKDCADPCRKQTGCPHAKCMNKTCRCH SEQ ID NO: 702GSVKCTTSKECWPPCKAATGKAAGKCMNKKCKCQ SEQ ID NO: 703GSLECGASRECYDPCFKAFGRAHGKCMNNKCRCY SEQ ID NO: 704GSEKCFATSQCWTPCKKAIGSLQSKCMNGKCKCY SEQ ID NO: 705GSVRCYASRECWEPCRRVTGSAQAKCQNNQCRCY SEQ ID NO: 706GSVKCSASRECWVACKKVTGSGQGKCQNNQCRCY SEQ ID NO: 707GSVKCISSQECWIACKKVTGRFEGKCQNRQCRCY SEQ ID NO: 708GSVRCYDSRQCWIACKKVTGSTQGKCQNKQCRCY SEQ ID NO: 709GSVDCTVSKECWAPCKAAFGVDRGKCMGKKCKCY SEQ ID NO: 710GSAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCY SEQ ID NO: 711GSKKCQGGSCASVCRRVIGVAAGKCINGRCVCY SEQ ID NO: 712GSKKCSNTSQCYKTCEKVVGVAAGKCMNGKCICY SEQ ID NO: 713GSVKCSGSSKCVKICIDRYNTRGAKCINGRCTCY SEQ ID NO: 714GSNRCNNSSECIPHCIRIFGTRAAKCINRKCYCY SEQ ID NO: 715GSKECNGSSECYSHCEGITGKRSGKCINKKCYCY SEQ ID NO: 716GSAFCNLRRCELSCRSLGLLGKCIGEECKCV SEQ ID NO: 717GSAVCNLKRCQLSCRSLGLLGKCIGDKCECV SEQ ID NO: 718GSAACYSS-DCRVKCVAMGFSSGKCINSKCKCY SEQ ID NO: 719GSAICATDADCSRKCPGNPPCRNGFCACT SEQ ID NO: 720GSTECQIKNDCQRYCQSVKECKYGKCYCN SEQ ID NO: 721GSTQCQSVRDCQQYCLTPDRCSYGTCYCK SEQ ID NO: 722GSVSCRYGSDCAEPCKRLKCLLPSKCINGKCTCY SEQ ID NO: 723GSIKCRYPADCHIMCRKVTGRAEGKCMNGKCTCY SEQ ID NO: 724GSIKCSSSSSCYEPCRGVTGRAHGKCMNGRCTCY SEQ ID NO: 725GSVKCTGSKQCLPACKAAVGKAAGKCMNGKCKCY SEQ ID NO: 726GSVSCKHSGQCIKPCKDA-GMRFGKCMNRKCDCT SEQ ID NO: 727GSVKCRGSPQCIQPCRDA-GMRFGKCMNGKCHCT SEQ ID NO: 728GSVKCTSPKQCLPPCKAQFGIRAGAKCMNGKCKCY SEQ ID NO: 729GSVKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCY SEQ ID NO: 730GSVKCTSPKQCLPPCKEIYGRHAGAKCMNGKCHCS SEQ ID NO: 731GSVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCY SEQ ID NO: 732GSVKCRGSRDCLDPCKKAGMRFGKCINSKCHCT SEQ ID NO: 733GSVRCVTDDDCFRKCPGNPSCKRGFCACK SEQ ID NO: 734GSVPCNNSRPCVPVCIREVNNKNGKCSNGKCLCY SEQ ID NO: 735GSVPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP SEQ ID NO: 736GSVQTNVKCQGGSCASVCRREIGVAAGKCINGKCVCYRN SEQ ID NO: 737GSAEIIRCSGTRECYAPCQKLTGCLNAKCMNKACKCYGCV SEQ ID NO: 738GSRPTDIKCSASYQCFPVCKSRFGKTNGRCVNGLCDCF SEQ ID NO: 739GSQFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS SEQ ID NO: 740GSVGINVKCKHSRQCLKPCKDAGMRFGKCTNGKCHCTPK SEQ ID NO: 741GSVVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDC SEQ ID NO: 742GSNFKVEGACSKPCRKYCIDKGARNGKCINGRCHCYY SEQ ID NO: 743GSQIDTNVKCSGSSKCVKICIDRYNTRGAKCINGRCTCYP SEQ ID NO: 744GSGVPISVRCRGSRDCLEPCRRAGTRFGRCINGRCHCTP SEQ ID NO: 745GSGVPISVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 746GSGVPISVRCRGSRDCLEPCRRAGTRFGRCINRRCHCTP SEQ ID NO: 747GSGVPINVRCRGSRDCLEPCRRAGTRFGRCINSRCHCTP SEQ ID NO: 748GSGVPINVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 749GSGVPINVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCYP SEQ ID NO: 750GSGVPINVRCRGSRDCYEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 751GSGVPINVRCRGSRDCLEPCRRAGTRFGRCIQSRCYCTP SEQ ID NO: 752GSGVPISVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCYP SEQ ID NO: 753GSGVPISVRCRGSRDCYEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 754GSGVPISVRCRGSRDCLEPCRRAGTRFGRCIQSRCYCTP SEQ ID NO: 755GSGVPINVRCRGSRDCLEPCRRAGTRFGRCIASRCHCYP SEQ ID NO: 756GSGVPINVRCRGSRDCLEPCRRAGTRFGRCISSRCHCYP SEQ ID NO: 757GSGVPINVRCRGSRDCLEPCRRAGTRFGRCITSRCHCYP SEQ ID NO: 758GSGVPINVRCRGSRDCLEPCRRAGTRFGRCINSRCHCYP SEQ ID NO: 798GIVCKVCKIICGMQGKKVNICKAPIKCKCKKG SEQ ID NO: 799SEKDCIKHLQRCRENKDCCSKKCSRRGTNPEKRCR SEQ ID NO: 800VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK SEQ ID NO: 801GVPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP SEQ ID NO: 802AVCVYRTCDKDCKRRGYRSGKCINNACKCYPYG SEQ ID NO: 803ISCTGSKQCYDPCKRKTGCPNAKCMNKSCKCYGCG SEQ ID NO: 804QVQTNVKCQGGSCASVCRREIGVAAGKCINGKCVCYRN SEQ ID NO: 805EVIRCSGSKQCYGPCKQQTGCTNSKCMNKVCKCYGCG SEQ ID NO: 806ACKGVFDACTPGKNECCPNRVCSDKHKWCKWKL SEQ ID NO: 807QIYTSKECNGSSECYSHCEGITGKRSGKCINKKCYCYR SEQ ID NO: 808GCLEFWWKCNPNDDKCCRPKLKCSKLFKLCNFSFG SEQ ID NO: 809DCVRFWGKCSQTSDCCPHLACKSKWPRNICVWDGSVG SEQ ID NO: 810GCFGYKCDYYKGCCSGYVCSPTWKWCVRPGPGR SEQ ID NO: 811MNAKFILLLVLTTMMLLPDTKGAEVIRCSGSKQCYGPCKQQTGCTNSKCM NKVCKCYGCGSEQ ID NO: 812 MNAKLIYLLLVVTTMTLMFDTAQAVDIMCSGPKQCYGPCKKETGCPNAKCMNRRCKCYGCV SEQ ID NO: 813MNAKLIYLLLVVTTMMLTFDTTQAGDIKCSGTRQCWGPCKKQTTCTNSKC MNGKCKCYGCVGSEQ ID NO: 814 MNTKFIFLLLVVTNTMMLFDTKPVEGISCTGSKQCYDPCKRKTGCPNAKCMNKSCKCYGCG SEQ ID NO: 815 GVPINVKCSGSRDCLEPCKKAGMRFGKCINRKCHCTPKSEQ ID NO: 816 GVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHCTPK SEQ ID NO: 817GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK SEQ ID NO: 818GVPINVKCRGSPQCIQPCRDAGMRFGKCMNGKCHCTPQ SEQ ID NO: 819GVEINVKCTGSHQCIKPCKDAGMRFGKCINRKCHCTPK SEQ ID NO: 820GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK SEQ ID NO: 821GVPTDVKCRGSPQCIQPCKDAGMRFGKCMNGKCHCTPK SEQ ID NO: 822GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK SEQ ID NO: 823GVPINVPCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK SEQ ID NO: 824VGINVKCKHSGQCLKPCKDAGMRFGKCINGKCDCTPK SEQ ID NO: 825VGINVKCKHSGQCLKPCKDAGMRFGKCMNGKCDCTPK SEQ ID NO: 826VGIPVSCKHSGQCIKPCKDAGMRFGKCMNRKCDCTPK SEQ ID NO: 827RKGCFKEGHSCPKTAPCCRPLVCKGPSPNTKKCTRP SEQ ID NO: 828SFCIPFKPCKSDENCCKKFKCKTTGIVKLCRW SEQ ID NO: 829LKGCLPRNRFCNALSGPRCCSGLRCKELSIWASKCL SEQ ID NO: 830GNYCLRGRCLPGGRKCCNGRPCECFAKICSCKPK SEQ ID NO: 831TVKCGGCNRKCCPGGCRSGKCINGKCQCY SEQ ID NO: 832GCMKEYCAGQCRGKVSQDYCLKHCKCIPR SEQ ID NO: 833ACLGFGEKCNPSNDKCCKSSSLVCSQKHKWCKYG SEQ ID NO: 834RGGCLPHNRFCNALSGPRCCSGLRCKELSIRDSRCLG SEQ ID NO: 835RGGCLPRNKFCNPSSGPRCCSGLTCKELNIWASKCL SEQ ID NO: 836QRSCAKPGDMCMGIKCCDGQCGCNRGTGRCFCK SEQ ID NO: 837ARGCADAYKSCNHPRTCCDGYNGYKRACICSGSNCKCKKS SEQ ID NO: 838RGGCLPHNRFCNALSGPRCCSGLRCKELSIWDSRCLG SEQ ID NO: 839RGGCLPHNRFCNALSGPRCCSGLKCKELSIYDSRCLG SEQ ID NO: 840RGGCLPHNRFCNALSGPRCCSRLKCKELSIWDSRCLG SEQ ID NO: 841RGGCLPHNRFCNALTGPRCCSRLRCKELSIWDSICLG SEQ ID NO: 842SCADAYKSCDSLKCCNNRTCMCSMIGTNCTCRKK SEQ ID NO: 843ERRCLPAGKTCVRGPMRVPCCGSCSQNKCT SEQ ID NO: 844LCSREGEFCYKLRKCCAGFYCKAFVLHCYRN SEQ ID NO: 845 ACGSCRKKCKGSGKCINGRCKCYSEQ ID NO: 846 ACGSCRKKCKGPGKCINGRCKCY SEQ ID NO: 847ACQGYMRKCGRDKPPCCKKLECSKTWRWCVWN SEQ ID NO: 848GRYCQKWMWTCDSKRACCEGLRCKLWCRKI SEQ ID NO: 849NAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP SEQ ID NO: 850NVKCRGSKECLPACKAAVGKAAGKCMNGKCKCYP SEQ ID NO: 851NVKCRGSPECLPKCKEAIGKSAGKCMNGKCKCYP SEQ ID NO: 852NAKCRGSPECLPKCKQAIGKAAGKCMNGKCKCYP SEQ ID NO: 853RGYCAEKGIKCHNIHCCSGLTCKCKGSSCVCRK SEQ ID NO: 854ERGCKLTFWKCKNKKECCGWNACALGICMPR SEQ ID NO: 855KKKCIAKDYGRCKWGGTPCCRGRGCICSIMGTNCECKPR SEQ ID NO: 856GCKLTFWKCKNKKECCGWNACALGICMPR SEQ ID NO: 857ACKGLFVTCTPGKDECCPNHVCSSKHKWCKYK SEQ ID NO: 858IACAPRGLLCFRDKECCKGLTCKGRFVNTWPTFCLV SEQ ID NO: 859ACAGLYKKCGKGVNTCCENRPCKCDLAMGNCICKKK SEQ ID NO: 860FTCAISCDIKVNGKPCKGSGEKKCSGGWSCKFNVCVKV SEQ ID NO: 861GFCAQKGIKCHDIHCCTNLKCVREGSNRVCRKA SEQ ID NO: 862CAKKRNWCGKNEDCCCPMKCIYAWYNQQGSCQSTITGLFKKC SEQ ID NO: 863YCQKWMWTCDSARKCCEGLVCRLWCKKI SEQ ID NO: 864RGGCLPHNKFCNALSGPRCCSGLKCKELTIWNTKCLE SEQ ID NO: 865NVKCTGSKQCLPACKAAVGKAAGKCMNGKCKCYT SEQ ID NO: 866QRSCAKPGEMCMRIKCCDGQCGCNRGTGRCFCK SEQ ID NO: 867GCIPKHKRCTWSGPKCCNNISCHCNISGTLCKCRPG SEQ ID NO: 868NYCVAKRCRPGGRQCCSGKPCACVGKVCKCPRD SEQ ID NO: 869ERGCSGAYKRCSSSQRCCEGRPCVCSAINSNCKCRKT SEQ ID NO: 870RYCPRNPEACYNYCLRTGRPGGYCGGRSRITCFCFR SEQ ID NO: 871QRSCAKPGEMCMGIKCCDGQCGCNRGTGRCFCK SEQ ID NO: 872RRGCFKEGKWCPKSAPCCAPLKCKGPSIKQQKCVRE SEQ ID NO: 873TVKCGGCNRKCCAGGCRSGKCINGKCQCYGR SEQ ID NO: 874ERRCEPSGKPCRPLMRIPCCGSCVRGKCA SEQ ID NO: 875RGGCLPRNKFCNPSSGPRCCSGLTCKELNIWANKCL SEQ ID NO: 876CAKKRNWCGKNEDCCCPMKCIYAWYNQQGSCQTTITGLFKKC SEQ ID NO: 877VRIPVSCKHSGQCLKPCKDAGMRTGKCMNGKCDCTPK SEQ ID NO: 878VKCTTSKDCWPPCKKVTGRA SEQ ID NO: 879 GIVCRVCRIICGMQGRRVNICRAPIRCRCRRGSEQ ID NO: 880 SERDCIRHLQRCRENRDCCSRRCSRRGTNPERRCR SEQ ID NO: 881VRIPVSCRHSGQCLRPCRDAGMRFGRCMNGRCDCTPR SEQ ID NO: 882GVPINVRCRGSRDCLDPCRRAGMRFGRCINSRCHCTP SEQ ID NO: 883AVCVYRTCDRDCRRRGYRSGRCINNACRCYPYG SEQ ID NO: 884ISCTGSRQCYDPCRRRTGCPNARCMNRSCRCYGCG SEQ ID NO: 885QVQTNVRCQGGSCASVCRREIGVAAGRCINGRCVCYRN SEQ ID NO: 886EVIRCSGSRQCYGPCRQQTGCTNSRCMNRVCRCYGCG SEQ ID NO: 887ACRGVFDACTPGRNECCPNRVCSDRHRWCRWRL SEQ ID NO: 888QIYTSRECNGSSECYSHCEGITGRRSGRCINRRCYCYR SEQ ID NO: 889GCLEFWWRCNPNDDRCCRPRLRCSRLFRLCNFSFG SEQ ID NO: 890DCVRFWGRCSQTSDCCPHLACRSRWPRNICVWDGSVG SEQ ID NO: 891GCFGYRCDYYRGCCSGYVCSPTWRWCVRPGPGR SEQ ID NO: 892MNARFILLLVLTTMMLLPDTRGAEVIRCSGSRQCYGPCRQQTGCTNSRCM NRVCRCYGCGSEQ ID NO: 893 MNARLIYLLLVVTTMTLMFDTAQAVDIMCSGPRQCYGPCRRETGCPNARCMNRRCRCYGCV SEQ ID NO: 894MNARLIYLLLVVTTMMLTFDTTQAGDIRCSGTRQCWGPCRRQTTCTNSRC MNGRCRCYGCVGSEQ ID NO: 895 MNTRFIFLLLVVTNTMMLFDTRPVEGISCTGSRQCYDPCRRRTGCPNARCMNRSCRCYGCG SEQ ID NO: 896 GVPINVRCSGSRDCLEPCRRAGMRFGRCINRRCHCTPRSEQ ID NO: 897 GVPINVRCTGSPQCLRPCRDAGMRFGRCINGRCHCTPR SEQ ID NO: 898GVIINVRCRISRQCLEPCRRAGMRFGRCMNGRCHCTPR SEQ ID NO: 899GVPINVRCRGSPQCIQPCRDAGMRFGRCMNGRCHCTPQ SEQ ID NO: 900GVEINVRCTGSHQCIRPCRDAGMRFGRCINRRCHCTPR SEQ ID NO: 901GVEINVRCSGSPQCLRPCRDAGMRFGRCMNRRCHCTPR SEQ ID NO: 902GVPTDVRCRGSPQCIQPCRDAGMRFGRCMNGRCHCTPR SEQ ID NO: 903GVPINVSCTGSPQCIRPCRDAGMRFGRCMNRRCHCTPR SEQ ID NO: 904GVPINVPCTGSPQCIRPCRDAGMRFGRCMNRRCHCTPR SEQ ID NO: 905VGINVRCRHSGQCLRPCRDAGMRFGRCINGRCDCTPR SEQ ID NO: 906VGINVRCRHSGQCLRPCRDAGMRFGRCMNGRCDCTPR SEQ ID NO: 907VGIPVSCRHSGQCIRPCRDAGMRFGRCMNRRCDCTPR SEQ ID NO: 908RRGCFREGHSCPRTAPCCRPLVCRGPSPNTRRCTRP SEQ ID NO: 909SFCIPFRPCRSDENCCRRFRCRTTGIVRLCRW SEQ ID NO: 910LRGCLPRNRFCNALSGPRCCSGLRCRELSIWASRCL SEQ ID NO: 911GNYCLRGRCLPGGRRCCNGRPCECFARICSCRPR SEQ ID NO: 912TVRCGGCNRRCCPGGCRSGRCINGRCQCY SEQ ID NO: 913GCMREYCAGQCRGRVSQDYCLRHCRCIPR SEQ ID NO: 914ACLGFGERCNPSNDRCCRSSSLVCSQRHRWCRYG SEQ ID NO: 915RGGCLPHNRFCNALSGPRCCSGLRCRELSIRDSRCLG SEQ ID NO: 916RGGCLPRNRFCNPSSGPRCCSGLTCRELNIWASRCL SEQ ID NO: 917QRSCARPGDMCMGIRCCDGQCGCNRGTGRCFCR SEQ ID NO: 918ARGCADAYRSCNHPRTCCDGYNGYRRACICSGSNCRCRRS SEQ ID NO: 919RGGCLPHNRFCNALSGPRCCSGLRCRELSIWDSRCLG SEQ ID NO: 920RGGCLPHNRFCNALSGPRCCSGLRCRELSIYDSRCLG SEQ ID NO: 921RGGCLPHNRFCNALSGPRCCSRLRCRELSIWDSRCLG SEQ ID NO: 922RGGCLPHNRFCNALTGPRCCSRLRCRELSIWDSICLG SEQ ID NO: 923SCADAYKSCDSLRCCNNRTCMCSMIGTNCTCRRR SEQ ID NO: 924ERRCLPAGRTCVRGPMRVPCCGSCSQNRCT SEQ ID NO: 925LCSREGEFCYRLRRCCAGFYCRAFVLHCYRN SEQ ID NO: 926 ACGSCRRRCRGSGRCINGRCRCYSEQ ID NO: 927 ACGSCRRRCRGPGRCINGRCRCY SEQ ID NO: 928ACQGYMRRCGRDRPPCCRRLECSRTWRWCVWN SEQ ID NO: 929GRYCQRWMWTCDSRRACCEGLRCRLWCRRI SEQ ID NO: 930NARCRGSPECLPRCREAIGRAAGRCMNGRCRCYP SEQ ID NO: 931NVRCRGSRECLPACRAAVGRAAGRCMNGRCRCYP SEQ ID NO: 932NVRCRGSPECLPRCREAIGRSAGRCMNGRCRCYP SEQ ID NO: 933NARCRGSPECLPRCRQAIGRAAGRCMNGRCRCYP SEQ ID NO: 934RGYCAERGIRCHNIHCCSGLTCRCRGSSCVCRR SEQ ID NO: 935ERGCRLTFWRCRNRRECCGWNACALGICMPR SEQ ID NO: 936RRRCIARDYGRCRWGGTPCCRGRGCICSIMGTNCECRPR SEQ ID NO: 937GCRLTFWRCRNRRECCGWNACALGICMPR SEQ ID NO: 938ACRGLFVTCTPGRDECCPNHVCSSRHRWCRYR SEQ ID NO: 939IACAPRGLLCFRDRECCRGLTCRGRFVNTWPTFCLV SEQ ID NO: 940ACAGLYRRCGRGVNTCCENRPCRCDLAMGNCICRRR SEQ ID NO: 941FTCAISCDIRVNGRPCRGSGERRCSGGWSCRFNVCVRV SEQ ID NO: 942GFCAQRGIRCHDIHCCTNLRCVREGSNRVCRRA SEQ ID NO: 943CARRRNWCGRNEDCCCPMRCIYAWYNQQGSCQSTITGLFRRC SEQ ID NO: 944YCQRWMWTCDSARRCCEGLVCRLWCRRI SEQ ID NO: 945RGGCLPHNRFCNALSGPRCCSGLRCRELTIWNTRCLE SEQ ID NO: 946NVRCTGSRQCLPACRAAVGRAAGRCMNGRCRCYT SEQ ID NO: 947QRSCARPGEMCMRIRCCDGQCGCNRGTGRCFCR SEQ ID NO: 948GCIPRHRRCTWSGPRCCNNISCHCNISGTLCRCRPG SEQ ID NO: 949NYCVARRCRPGGRQCCSGRPCACVGRVCRCPRD SEQ ID NO: 950ERGCSGAYRRCSSSQRCCEGRPCVCSAINSNCRCRRT SEQ ID NO: 951QRSCARPGEMCMGIRCCDGQCGCNRGTGRCFCR SEQ ID NO: 952RRGCFREGRWCPRSAPCCAPLRCRGPSIRQQRCVRE SEQ ID NO: 953TVRCGGCNRRCCAGGCRSGRCINGRCQCYGR SEQ ID NO: 954ERRCEPSGRPCRPLMRIPCCGSCVRGRCA SEQ ID NO: 955RGGCLPRNRFCNPSSGPRCCSGLTCRELNIWANRCL SEQ ID NO: 956CARRRNWCGRNEDCCCPMRCIYAWYNQQGSCQTTITGLFRRC SEQ ID NO: 957VRIPVSCRHSGQCLRPCRDAGMRTGRCMNGRCDCTPR SEQ ID NO: 958QKILSNRCNNSSECIPHCIRIFGTRAAKCINRKCYCYP SEQ ID NO: 959AVCNLKRCQLSCRSLGLLGKCIGDKCECVKHG SEQ ID NO: 960ISIGIRCSPSIDLCEGQCRIRRYFTGYCSGDTCHCSG SEQ ID NO: 961GDCLPHLRRCRENNDCCSRRCRRRGANPERRCR SEQ ID NO: 962SCEPGRTFRDRCNTCKCGADGRSAACTLRACPNQ SEQ ID NO: 963GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR SEQ ID NO: 964GDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR SEQ ID NO: 965KDCLKKLKLCKENKDCCSKSCKRRGTNIEKRCR SEQ ID NO: 966GDCLPHLKRCKENNDCCSKKCKRRGANPEKRCR SEQ ID NO: 967VFINVKCRGSPECLPKCKEAIGKSAGKCMNGKCKCYP SEQ ID NO: 968VFINAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP SEQ ID NO: 969VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP SEQ ID NO: 970VPTDVKCRGSPQCIQPCKDAGMRFGKCMNGKCHCTP SEQ ID NO: 971VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDCTP SEQ ID NO: 972VRIPVSCRHSGQCLRPCRDAGMRFGRCMNGRCDCTP SEQ ID NO: 973TNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRC SEQ ID NO: 974NVKCTGSKQCLPACKAAVGKAAGKCMNGKCKC SEQ ID NO: 975GVPINVRCRGSRDCLDPCRGAGERHGRCGNSRCHCTP SEQ ID NO: 976VRIPVSCRHSGQCLRPCRDAGERHGRCGGGRCDCTPR SEQ ID NO: 977QVQTNVRCQGGSCGSVCRREGGGAGGGCGNGRCGCYRN SEQ ID NO: 978IKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF SEQ ID NO: 979VKCSSPQQCLKPCKAAFGISAGgKCINGKCKCY SEQ ID NO: 980VSCSASSQCWPVCKKLFGTYRGKCMNSKCRCY SEQ ID NO: 981ESCTASNQCWSICKRLHNTNRGKCMNKKCRCY SEQ ID NO: 982VSCTTSKECWSVCEKLYNTSRGKCMNKKCRCY SEQ ID NO: 983MRCKSSKECLVKCKQATGRPNGKCMNRKCKCY SEQ ID NO: 984IKCTLSKDCYSPCKKETGCPRAKCINRNCKCY SEQ ID NO: 985IRCSGSRDCYSPCMKQTGCPNAKCINKSCKCY SEQ ID NO: 986IRCSGTRECYAPCQKLTGCLNAKCMNKACKCY SEQ ID NO: 987ISCTNPKQCYPHCKKETGYPNAKCMNRKCKCF SEQ ID NO: 988ASCRTPKDCADPCRKETGCPYGKCMNRKCKCN SEQ ID NO: 989TSCISPKQCTEPCRAKGCKHGKCMNRKCHCM SEQ ID NO: 990KECTGPQHCTNFCRKN-KCTHGKCMNRKCKCF SEQ ID NO: 991IKCRTPKDCADPCRKQTGCPHAKCMNKTCRCH SEQ ID NO: 992VKCTTSKECWPPCKAATGKAAGKCMNKKCKCQ SEQ ID NO: 993LECGASRECYDPCFKAFGRAHGKCMNNKCRCY SEQ ID NO: 994EKCFATSQCWTPCKKAIGSLQSKCMNGKCKCY SEQ ID NO: 995VRCYASRECWEPCRRVTGSAQAKCQNNQCRCY SEQ ID NO: 996VKCSASRECWVACKKVTGSGQGKCQNNQCRCY SEQ ID NO: 997VKCISSQECWIACKKVTGRFEGKCQNRQCRCY SEQ ID NO: 998VRCYDSRQCWIACKKVTGSTQGKCQNKQCRCY SEQ ID NO: 999VDCTVSKECWAPCKAAFGVDRGKCMGKKCKCY SEQ ID NO: 1000AKCRGSPECLPKCKEAIGKAAGKCMNGKCKCY SEQ ID NO: 1001KKCQGGSCASVCRRVIGVAAGKCINGRCVCY SEQ ID NO: 1002KKCSNTSQCYKTCEKVVGVAAGKCMNGKCICY SEQ ID NO: 1003VKCSGSSKCVKICIDRYNTRGAKCINGRCTCY SEQ ID NO: 1004NRCNNSSECIPHCIRIFGTRAAKCINRKCYCY SEQ ID NO: 1005KECNGSSECYSHCEGITGKRSGKCINKKCYCY SEQ ID NO: 1006AFCNLRRCELSCRSLGLLGKCIGEECKCV SEQ ID NO: 1007AVCNLKRCQLSCRSLGLLGKCIGDKCECV SEQ ID NO: 1008AACYSS-DCRVKCVAMGFSSGKCINSKCKCY SEQ ID NO: 1009AICATDADCSRKCPGNPPCRNGFCACT SEQ ID NO: 1010 TECQIKNDCQRYCQSVKECKYGKCYCNSEQ ID NO: 1011 TQCQSVRDCQQYCLTPDRCSYGTCYCK SEQ ID NO: 1012VSCRYGSDCAEPCKRLKCLLPSKCINGKCTCY SEQ ID NO: 1013IKCRYPADCHIMCRKVTGRAEGKCMNGKCTCY SEQ ID NO: 1014IKCSSSSSCYEPCRGVTGRAHGKCMNGRCTCY SEQ ID NO: 1015VKCTGSKQCLPACKAAVGKAAGKCMNGKCKCY SEQ ID NO: 1016VSCKHSGQCIKPCKDA-GMRFGKCMNRKCDCT SEQ ID NO: 1017VKCRGSPQCIQPCRDA-GMRFGKCMNGKCHCT SEQ ID NO: 1018VKCTSPKQCLPPCKAQFGIRAGAKCMNGKCKCY SEQ ID NO: 1019VKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCY SEQ ID NO: 1020VKCTSPKQCLPPCKEIYGRHAGAKCMNGKCHCS SEQ ID NO: 1021VKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCY SEQ ID NO: 1022VKCRGSRDCLDPCKKAGMRFGKCINSKCHCT SEQ ID NO: 1023VRCVTDDDCFRKCPGNPSCKRGFCACK SEQ ID NO: 1024VPCNNSRPCVPVCIREVNNKNGKCSNGKCLCY SEQ ID NO: 1025VPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP SEQ ID NO: 1026VQTNVKCQGGSCASVCRREIGVAAGKCINGKCVCYRN SEQ ID NO: 1027AEIIRCSGTRECYAPCQKLTGCLNAKCMNKACKCYGCV SEQ ID NO: 1028RPTDIKCSASYQCFPVCKSRFGKTNGRCVNGLCDCF SEQ ID NO: 1029QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS SEQ ID NO: 1030VGINVKCKHSRQCLKPCKDAGMRFGKCTNGKCHCTPK SEQ ID NO: 1031VVIGQRCYRSPDCYSACKKLVGKATGKCTNGRCDC SEQ ID NO: 1032NFKVEGACSKPCRKYCIDKGARNGKCINGRCHCYY SEQ ID NO: 1033QIDTNVKCSGSSKCVKICIDRYNTRGAKCINGRCTCYP SEQ ID NO: 1034GVPISVRCRGSRDCLEPCRRAGTRFGRCINGRCHCTP SEQ ID NO: 1035GVPISVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 1036GVPISVRCRGSRDCLEPCRRAGTRFGRCINRRCHCTP SEQ ID NO: 1037GVPINVRCRGSRDCLEPCRRAGTRFGRCINSRCHCTP SEQ ID NO: 1038GVPINVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 1039GVPINVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCYP SEQ ID NO: 1040GVPINVRCRGSRDCYEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 1041GVPINVRCRGSRDCLEPCRRAGTRFGRCIQSRCYCTP SEQ ID NO: 1042GVPISVRCRGSRDCLEPCRRAGTRFGRCIQSRCHCYP SEQ ID NO: 1043GVPISVRCRGSRDCYEPCRRAGTRFGRCIQSRCHCTP SEQ ID NO: 1044GVPISVRCRGSRDCLEPCRRAGTRFGRCIQSRCYCTP SEQ ID NO: 1045GVPINVRCRGSRDCLEPCRRAGTRFGRCIASRCHCYP SEQ ID NO: 1046GVPINVRCRGSRDCLEPCRRAGTRFGRCISSRCHCYP SEQ ID NO: 1047GVPINVRCRGSRDCLEPCRRAGTRFGRCITSRCHCYP SEQ ID NO: 1048GVPINVRCRGSRDCLEPCRRAGTRFGRCINSRCHCYP

In any of SEQ ID NO: 485—SEQ ID NO: 1048 or fragment thereof, any one ormore K residues can be replaced by an R residue or an A residue, any oneor more R residues can be replaced by a K residue or an A residue, anyone or more A residues can be replaced by a K residue or an R residue,all K residues can be replaced by R residues or A residues, all but oneK residue can be replaced by R or A residues, all but two K residues canbe replaced by R residues or A residues, or in any combination thereof.In any of SEQ ID NO: 485—SEQ ID NO: 1048 or any fragment thereof, anyone or more M residues can be replaced by any one of I, L, or Vresidues, any one or more L residues can be replaced by any one of V, I,or M residues, any one or more I residues can be replaced by any one ofM, L, or V residues, or any one or more V residues can be replaced byany one of I, L, or M residues. In any embodiment, at least one of theamino acids alone or in combination can be interchanged in the peptidesor peptide fragments as follows: K/R, M/ I/ LN, G/A, S/T, Q/N, and D/Ewherein each letter is each individually any amino acid or amino acidanalogue. In some instances, the peptide can contain only one lysineresidue, or no lysine residue. In any of SEQ ID NO: 485—SEQ ID NO: 1048or fragment thereof, any amino acid can be replaced with citrulline. Inany of SEQ ID NO: 485—SEQ ID NO: 1048 or any fragment thereof, X canindependently be any number of any amino acid or no amino acid. In somecases, a peptide can include the first two N-terminal amino acids GS, aswith peptides of SEQ ID NO: 485—SEQ ID NO: 758, or such N-terminal aminoacids (GS) can be substituted by any other one or two amino acids. Inother cases, a peptide does not include the first two N-terminal aminoacids GS, as with peptides of SEQ ID NO: 759—SEQ ID NO: 1048. In somecases, the N-terminus of the peptide is blocked, such as by an acetylgroup; in other instances, the C-terminus of the peptide is blocked,such as by an amide group.

In some instances, the peptide is any one of SEQ ID NO: 485—SEQ ID NO:1048 or a functional fragment thereof. In other embodiments, the peptideof the disclosure further comprises a peptide with 100%, 99%, 97%, 95%,90%, 85%, or 80% homology to any one of SEQ ID NO: 485—SEQ ID NO: 1048.In further embodiments, the peptide fragment comprises a contiguousfragment of any one of SEQ ID NO: 485—SEQ ID NO: 1048 that is at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, at least 30, at least 31, at least 32, at least 33, atleast 34, at least 35, at least 36, at least 37, at least 38, at least39, at least 40, at least 41, at least 42, at least 43, at least 44, atleast 45, at least 46 residues long, wherein the peptide fragment isselected from any portion of the peptide. In some embodiments, suchpeptide fragments contact the cartilage and exhibit properties of thosedescribed herein for peptide and peptide-active agent conjugates.

The peptides of the present disclosure can further comprise negativeamino acid residues. In some cases, the peptide has 2 or fewer negativeamino acid residues. In other cases, the peptide has 4 or fewer negativeamino acid residues, 3 or fewer negative amino acid residues, or 1 orfewer negative amino acid residues. The negative amino acid residues canbe selected from any negative charged amino acid residues. The negativeamino acid residues can be selected from either E or D, or a combinationof both E and D.

The peptides of the present disclosure can further comprise basic aminoacid residues. In some embodiments, basic residues are added to thepeptide sequence to increase the charge at physiological pH. The addedbasic residues can be any basic amino acid. The added basic residues canbe selected from K or R, or a combination of K or R.

In some embodiments, the peptide has a charge distribution comprising anacidic region and a basic region. An acidic region can be a nub. A nubis a portion of a peptide extending out of the peptide'sthree-dimensional structure. A basic region can be a patch. A patch is aportion of a peptide that does not designate any specific topologycharacteristic of the peptide's three-dimensional structure. In furtherembodiments, a cystine-dense peptide can be 6 or more basic residues and2 or fewer acidic residues.

The peptides of the present disclosure can further comprise positivelycharged amino acid residues. In some cases, the peptide has at least 2positively charged residues. In other cases, the peptide has at least 3positively charged residues, at least 4 positively charged residues, atleast 5 positively charged residues, at least 6 positively chargedresidues, at least 7 positively charged residues, at least 8 positivelycharged residues or at least 9 positively charged residues. Thepositively charged residues can be selected from any positively chargedamino acid residues. The positively charged residues can be selectedfrom either K or R, or a combination of K and R.

In addition, the peptides herein can comprise a 4-19 amino acid residuefragment of any of the above sequences containing at least 2 cysteineresidues, and at least 2 or 3 positively charged amino acid residues(for example, arginine, lysine or histidine, or any combination ofarginine, lysine or histidine). In other embodiments, the peptidesherein is a 20-70 amino acid residue fragment of any of the abovesequences containing at least 2 cysteine residues, no more than 2 basicresidues, and at least 2 or 3 positively charged amino acid residues(for example, arginine, lysine or histidine, or any combination ofarginine, lysine or histidine). In some embodiments, such peptidefragments contact the cartilage and exhibit properties of thosedescribed herein for peptide and peptide-active agent conjugates.

In some embodiments, the peptide contains one or more disulfide bondsand has a positive net charge at neutral pH. At physiological pH,peptides can have a net charge, for example, of −5, −4, −3, −2, −1, 0,+1, +2, +3, +4, or +5. When the net charge is zero, the peptide can beuncharged or zwitterionic. In some instances, the peptide can have apositive charge at physiological pH. In some instances, the peptide canhave a charge ≥+2 at physiological pH, ≥+3.5 at physiological pH, ≥+4.5at physiological pH. In some embodiments, the peptide contains one ormore disulfide bonds and has a positive net charge at neutral pH wherethe net charge can be +0.5 or less than +0.5, +1 or less than +1, +1.5or less than +1.5, +2 or less than +2, +2.5 or less than +2.5, +3 orless than +3, +3.5 or less than +3.5, +4 or less than +4, +4.5 or lessthan +4.5, +5 or less than +5, +5.5 or less than +5.5, +6 or less than+6, +6.5 or less than +6.5, +7 or less than +7, +7.5 or less than +7.5,+8 or less than +8, +8.5 or less than +8.5, +9 or less than +9.5, +10 orless than +10. In some embodiments, the peptide has a negative netcharge at physiological pH where the net charge can be -0.5 or less than−0.5, −1 or less than −1, −1.5 or less than −1.5, −2 or less than −2,−2.5 or less than −2.5, −3 or less than −3, −3.5 or less than −3.5, −4or less than −4, −4.5 or less than −4.5, −5 or less than −5, −5.5 orless than −5.5, −6 or less than −6, −6.5 or less than −6.5, −7 or lessthan −7, −7.5 or less than −7.5, −8 or less than −8, −8.5 or less than−8.5, −9 or less than −9.5, −10 or less than −10. In some cases, theengineering of one or more mutations within a peptide yields a peptidewith an altered isoelectric point, charge, surface charge, or rheologyat physiological pH. Such engineering of a mutation to a peptide derivedfrom a scorpion or spider can change the net charge of the complex, forexample, by decreasing the net charge by 1, 2, 3, 4, or 5, or byincreasing the net charge by 1, 2, 3, 4, or 5. In such cases, theengineered mutation may facilitate the ability of the peptide to contactthe cartilage. Suitable amino acid modifications for improving therheology and potency of a peptide can include conservative ornon-conservative mutations. A peptide can comprises at most 1 amino acidmutation, at most 2 amino acid mutations, at most 3 amino acidmutations, at most 4 amino acid mutations, at most 5 amino acidmutations, at most 6 amino acid mutations, at most 7 amino acidmutations, at most 8 amino acid mutations, at most 9 amino acidmutations, at most 10 amino acid mutations, or another suitable numberas compared to the sequence of the venom or toxin that the peptide isderived from. In other cases, a peptide, or a functional fragmentthereof, comprises at least 1 amino acid mutation, at least 2 amino acidmutations, at least 3 amino acid mutations, at least 4 amino acidmutations, at least 5 amino acid mutations, at least 6 amino acidmutations, at least 7 amino acid mutations, at least 8 amino acidmutations, at least 9 amino acid mutations, at least 10 amino acidmutations, or another suitable number as compared to the sequence of thevenom or toxin that the peptide is derived from. In some embodiments,mutations can be engineered within a peptide to provide a peptide thathas a desired charge or stability at physiological pH.

Peptides can be mutated to add function or remove function. For example,peptides and peptide-conjugates of the present disclosure can be mutatedto retain, remove, or add the ability to bind to ion channels, or topromote agonizing or antagonizing ion channels, such as potassiumchannel binding that may occur with the peptide or peptide-conjugates(e.g., the potassium channel hERG). In some instances, it can beadvantageous to remove potassium channel binding from a peptide used fordelivery of an active agent. Mutations can include one or more N to S, Dto E, M to T, N to Q, N to A, N to S, N to T, N to L, S to G, and S to Ramino acid substitutions, or one or more L to Y, H to Y, and T to Yamino acid substitutions, or any combination of thereof, depending onwhether the variant is designed to retain function or to remove functionof binding to the ion channel In some embodiments the peptides andpeptide-drug conjugates of the present disclosure are mutated tominimize ion channel binding in order to minimize side effects orenhance the safety either in the target tissue or systemically.

In some embodiments, charge can play a role in cartilage homing. Theinteraction of a peptide of this disclosure in solution and in vivo canbe influenced by the isoelectric point (pI) of the peptide and/or the pHof the solution or the local environment it is in. The charge of apeptide in solution can impact the solubility of the protein as well asparameters such as biodistribution, bioavailability, and overallpharmacokinetics. Additionally, positively charged molecules caninteract with negatively charged molecules. Positively charged moleculessuch as the peptides disclosed herein can interact and bind withnegatively charged molecules such as the negatively chargedextracellular matrix molecules in the cartilage including hyaluranon andaggrecan. Positively charged residues can also interact with specificregions of other proteins and molecules, such as negatively chargedresidues of receptors or electronegative regions of an ion channel poreon cell surfaces. As such, the pI of a peptide can influence whether apeptide of this disclosure can efficiently home to cartilage.Identifying a correlation between pI and cartilage homing can be animportant strategy in identifying lead peptide candidates of the presentdisclosure. The pI of a peptide can be calculated using a number ofdifferent methods including the Expasy pI calculator and the Silleromethod. The Expasy pI can be determined by calculating pKa values ofamino acids as described in Bjellqvist et al., which were defined byexamining polypeptide migration between pH 4.5 to pH 7.3 in animmobilized pH gradient gel environment with 9.2M and 9.8M urea at 15°C. or 25° C_(max) (Bjellqvist et al. Electrophoresis. 14(10):1023-31(1993)). The Sillero method of calculating pI can involve the solutionof a polynomial equation and the individual pKas of each amino acid.This method does not use denaturing conditions (urea) (Sillero et al.179(2): 319-35 (1989)) Using these pI calculation methods andquantifying the cartilage to blood ratio of peptide signal afteradministration to a subject can be a strategy for identifying a trend orcorrelation in charge and cartilage homing. In some embodiments, apeptide with a pI above biological pH (—pH 7. 4) can exhibit efficienthoming to cartilage. In some embodiments, a peptide with a pI of atleast 8, at least 9, at least 10, or at least 11 can efficiently home tocartilage. In other embodiments, a peptide with a pI of 11-12 can homemost efficiently to cartilage. In certain embodiments, a peptide canhave a pI of about 9. In other embodiments, a peptide can have a pI of8-10. In some embodiments, more basic peptides can home more efficientlyto cartilage. In other embodiments, a high pI alone may not besufficient to cause cartilage homing of a peptide.

In some embodiments, the tertiary structure and electrostatics of apeptide of the disclosure can impact cartilage homing. Structuralanalysis or analysis of charge distribution can be a strategy to predictresidues important in biological function, such as cartilage homing. Forexample, several peptides of this disclosure that home to cartilage canbe grouped into a structural class defined herein as “hitchins,” and canshare the properties of disulfide linkages between C1-C₄, C2-C5, andC3-C6. The folding topologies of peptides linked through three disulfidelinkages (C₁-C₄, C2-C5, and C3-C6), can be broken down into structuralfamilies based on the three-dimensional arrangement of the disulfides.Some cystine-dense peptides have the C3-C6 disulfide linkage passingthrough the macrocycle formed by the C1-C₄ and C2-C5 disulfide linkages,hitchins have the C2-C5 disulfide linkage passing through the macrocycleformed by the C1-C₄ and C3-C6 disulfide linkages, and yet otherstructural families have the C1-C₄ disulfide linkage passing through themacrocycle formed by the C2-C5 and C3-C6 disulfide linkages. Variants of“hitchin” class peptides with preserved disulfide linkages at thesecysteine residues, primary sequence identity, and/or structural homologycan be a method of identifying or predicting other potential peptidecandidates that can home to cartilage. Additionally, members and relatedmembers of the calcin family of peptides can also home to cartilage,despite having a distinct tertiary structure from the “hitchin” class ofpeptides. Calcin peptides are structurally a subset of the cystine-densepeptides, with cystine-dense disulfide connectivity and topology, butare further classified on the basis of functioning to bind and activateryanodine receptors (RyRs). These receptors are calcium channels thatact to regulate the influx and efflux of calcium in muscle (Schwartz etal. Br J Pharmacol 157(3):392-403. (2009)). Variants of the calcinfamily of peptides with preserved key residues can be one way to predictpromising candidates that can home to cartilage. In some embodiments,structural analysis of a peptide of this disclosure can be determined byevaluating peptides for resistance to degradation in buffers withvarious proteases or reducing agents. Structural analysis of thedistribution of charge density on the surface of a peptide can also be astrategy for predicting promising candidates that can home to cartilage.Peptides with large patches of positive surface charge (when at pH 7.5)can home to cartilage.

The NMR solution structures, x-ray crystallography, or crystalstructures of related structural homologs can be used to informmutational strategies that can improve the folding, stability, andmanufacturability, while maintaining the ability of a peptide to home tocartilage. They can be used to predict the 3D pharmacophore of a groupof structurally homologous scaffolds, as well as to predict possiblegraft regions of related proteins to create chimeras with improvedproperties. For example, this strategy can be used to identify criticalamino acid positions and loops that can be used to design drugs withimproved properties or to correct deleterious mutations that complicatefolding and manufacturability for the peptides. These key amino acidpositions and loops can be retained while other residues in the peptidesequences can be mutated to improve, change, remove, or otherwise modifyfunction, homing, and activity of the peptide.

Additionally, the comparison of the primary sequences and the tertiarysequences of two or more peptides can be used to reveal sequence and 3Dfolding patterns that can be leveraged to improve the peptides and parseout the biological activity of these peptides. For example, comparingtwo different peptide scaffolds that home to cartilage can lead to theidentification of conserved pharmacophores that can guide engineeringstrategies, such as designing variants with improved folding properties.Important pharmacophore, for example, can comprise aromatic residues orbasic residues, which can be important for binding.

Improved peptides can also be engineered based upon immunogenicityinformation, such as immunogenicity information predicted by TEPITOPEand TEPITOPEpan. TEPITOPE is a computational approach which usesposition specific scoring matrix to provide prediction rules for whethera peptide will bind to 51 different HLA-DR alleles, and TEPITOPEpan ismethod that uses TEPITOPE to extrapolate from HLA-DR molecules withknown binding specificities to HLA-DR molecules with unknown bindingspecificities based on pocket similarity. For example, TEPITOPE andTEPITOPEpan can be used to determine immunogenicity of peptides thathome to cartilage. Immunogenicity information can also be predictedusing the program NetMHCII version 2. 3, which can determine thelikelihood that a sequence might be presented as an immunogenic peptidevia the major histocompatibility complex (MHC) presentation system ofantigen presenting cells (APCs). (Nielson, M et al. BMC Bioinformatics,8: 238 (2007); Nielsen, M. et al. BMC Bioinformatics, 10: 296 (2009)).This program can create an immunogenicity score by predicting thebinding of a peptide to MHC alleles. Strong binding alleles and weakbinding alleles in each major MHC allele group (DR, DQ, and DP) can betallied separately. The number of peptides of a specific length withinthe sequence (e.g., a ‘core’ peptide that can be nine residues long)that are immunogenic can also be tallied. Comparison of peptides or‘core’ peptides with high immunogenicity to peptides or ‘core’ peptideswith low immunogenicity can guide engineering strategies for designingvariants with decreased immunogenicity. Stronger binding peptides can bemore likely to generate an immune response in patient carrying thatgiven MHC alleles. Mutating stronger binding amino acids or peptides outof a peptide sequence can reduce the immunogenicity of the entirepeptide. Another aspect of immunogenicity, in addition to whether apeptide binds to a patient's MHC allele, can be whether the patient'simmune cells, such as a professional antigen presenting cells such as amacrophage, a B cell, or a dendritic cell, can process the peptide. Adendritic cell can take up a protein or peptide, and then can process apeptide, such as by cleaving to form a nine residue long peptide, whichthen can bind to the MHC and can be presented on the surface of thedendritic cell to the immune system's various T cells, including helperT cells and cytotoxic T cells, and thus can stimulate an immuneresponse. The processing can involve peptide bond cleavage by enzymesand disulfide bond reduction, and thus a peptide or protein that isresistant to enzymatic cleavage and/or reduction can be resistant toprocessing and subsequent MHC presentation to the immune system.Therefore, having a peptide or protein that is resistant to enzymaticcleavage and/or reduction can reduce its immunogenic potential.

Furthermore, multiple sequence alignment can also be used to informmutational strategies using previously identified sequences, and thusproviding a guide to making changes that would eliminate labile residuesand immunogenic regions of a peptide sequence. Peptides can be evaluatedfor residues of potential biochemical instability and regions ofpotential immunogenicity. Then, a residue that can allow for greaterpeptide stability at a certain location in a peptide can be identifiedfrom a multiple sequence alignment. For example, a specific residue canbe identified from a multiple sequence alignment as providing greaterstability for a peptide at position previously identified as a possiblerisk for a significant rate of deamidation, cleavage, degradation,oxidation, hydrolysis, isomerization, disulfide exchange, racemization,beta elimination, or aggregation. This information can then be used tocreate peptides with greater stability or reduced immunogenicity.

In addition to utilizing co-crystal x-ray structures, NMR solutionstructures, and mutagenesis studies, a multiple alignment of peptidesequences can be used to identify specific amino acids or regions ofhigh conservation that indicate an important interaction with a targetor receptor (e.g., binding to a potassium channel protein) or areimportant for folding and structure or other properties. Once theconserved amino acid or region is identified, then amino acidsreplacements can be determined that maintain the important properties ofthe peptide, such as maintenance of the structure, reduction inimmunogenicity, reduction in binding to an ion channel protein,increased stability, or any combination of thereof

The multiple sequence alignment can also identify possible locations toadd a tyrosine or tryptophan residue for spectrophotometric reporting.Incorporation of aromatic amino acids such as Tyrosine or Tryptophaninto a peptide such as SEQ ID NO: 592, which otherwise contains onlyamino acids of low UV absorbance at 280 nm, can be analyticallyadvantageous. Tyrosine and Tryptophan amino acids contain aromatic ringstructures. These residues have distinct absorption and emissionwavelengths and good quantum yields, as shown in TABLE 52, not presentin other amino acids. Both Tyrosine and Tryptophan can provide a good‘handle’ for analytical detection of a peptide in solution since UVabsorbance in the 250-300 nm range and peptide fluorescence is specificfor these aromatic molecules. While detection of a peptide such as SEQID NO: 592 relies on the absorbance of the peptide bond at 220 nm, wheremany other materials including minor impurities in solvents also oftencontribute to signal, the absorbance and fluorescence properties ofTryptophan and Tyrosine containing peptides can provide for asignificantly more selective and sensitive detection. Thus incorporatingan aromatic amino acid can create peptides better suited forconcentration and purity measurements, which can be useful duringanalytics, process development, manufacturing, and other drugdevelopment and drug manufacturing activities. Incorporation can beachieved either through substitutions of one or more amino acids in thepeptide to Tyr and/or Trp, insertion of Tyr and/or Trp into the peptide,or via addition of Tyr and/or Trp to the N-terminus or C-terminus of thepeptide.

TABLE 52 Absorbance and Fluorescence Characteristics of Tryptophan andTyrosine. Absorbance Fluorescence Amino Wavelength AbsorbtivityWavelength Quantum Acid (nm) (M * cm)⁻¹ (nm) Yield Tryptophan 280 5,600348 0.20 Tyrosine 274 1,400 303 0.14

A peptide of this disclosure can bind to chloride, potassium, or sodiumchannels. The peptide can also bind to calcium channels. The peptide canblock potassium channels and/or sodium channels. The peptide can blockcalcium channels. In some embodiments, the peptide can activate any oneor more of such channels. In some embodiments, the peptide can block anyone or more of such channels. In some embodiments, the peptide cannotinteract with any of such channels or can be mutated to reduce or removebinding to any such channels. In still other embodiments, the peptidecan be a potassium channel agonist, a potassium channel antagonist, aportion of a potassium channel, a sodium channel agonist, a sodiumchannel antagonist, a chloride channel agonist, a chloride channelantagonist, a calcium channel agonist, a calcium channel antagonist, ahadrucalcin, a theraphotoxin, a huwentoxin, a kaliotoxin, a cobatoxin ora lectin. In some embodiments, the lectin can be SHL-Ib2. In someembodiments, the peptide can interact with, binds, inhibits,inactivates, or alters expression of ion channels or chloride channels.In some embodiments, the peptide can interact with an Nav1.7 ion channelIn some embodiments, the peptide can interact with a Kv 1.3 ion channelIn still other embodiments, the peptide interacts with proteases, matrixmetalloproteinase, inhibits cancer cell migration or metastases, hasantimicrobial activity, or has antitumor activity. In addition to actingon matrix metalloproteinases, the peptide can interact with otherpossible proteases (e.g., elastases). In some embodiments, a peptide ofthis disclosure can bind to multidrug resistance transporters. Peptideand peptide drug conjugate binding to and blocking multidrug resistancetransporters can be used to treat bacterial infections or cancers of thejoint and/or bone.

In some embodiments, the peptide has other therapeutic effects on thecartilage or structures thereof or nearby. Beta defensin expression inarticular cartilage can be correlated with immunomodulatory functions aswe well as osteoarthritis, autoimmune rheumatic disorders such assystemic lupus erythematosus and rheumatoid arthritis (Vordenbäumen andSchneider 2011, Varoga 2004 and Varoga 2005). In some embodiments, thepeptides or their mutants inhibit beta defensins, supplement betadefensins, are competitive inhibitors of beta defensins, active or blockactivation of beta defensin targets, and are used as immune modulators,or to treat autoimmune, arthritis, infections, and other articulardisorders.

The present disclosure can also encompass multimers of the variouspeptides described herein. Examples of multimers include dimers,trimers, tetramers, pentamers, hexamers, heptamers, and so on. Amultimer can be a homomer formed from a plurality of identical subunitsor a heteromer formed from a plurality of different subunits. In someembodiments, a peptide of the present disclosure is arranged in amultimeric structure with at least one other peptide, or two, three,four, five, six, seven, eight, nine, ten, or more other peptides. Incertain embodiments, the peptides of a multimeric structure each havethe same sequence. In alternative embodiments, some or all of thepeptides of a multimeric structure have different sequences.

The present disclosure further includes peptide scaffolds that, e.g.,can be used as a starting point for generating additional peptides. Insome embodiments, these scaffolds can be derived from a variety ofcystine-dense peptides. Some suitable peptides for scaffolds caninclude, but are not limited to, chlorotoxin, brazzein, circulin,stecrisp, hanatoxin, midkine, hefutoxin, potato carboxypeptidaseinhibitor, bubble protein, attractin, α-GI, α-GID, μ-PIIIA, ω-MVIIA,ω-CVID, χ-MrIA, ρ-TIA, conantokin G, contulakin G, GsMT×4, margatoxin,shK, toxin K, chymotrypsin inhibitor (CTI), and EGF epiregulin core.

In some embodiments, the peptide sequences of the disclosure are flankedby additional amino acids. One or more additional amino acids can, forexample, confer a desired in vivo charge, isoelectric point, chemicalconjugation site, stability, or physiologic property to a peptide.

Identifying sequence homology can be important for determining keyresidues that preserve cartilage homing function. For example, in someembodiments identification of conserved positively charged residues canbe important in preserving cartilage homing in any homologous variantsthat are made. In other embodiments, identification of basic or aromaticdyads, can be important in preserving interaction and activity with Kvion channels in homologous variants.

Two or more peptides can share a degree of homology and share similarproperties in vivo. For instance, a peptide can share a degree ofhomology with a peptide of the present disclosure. In some cases, apeptide of the disclosure can have up to about 20% pairwise homology, upto about 25% pairwise homology, up to about 30% pairwise homology, up toabout 35% pairwise homology, up to about 40% pairwise homology, up toabout 45% pairwise homology, up to about 50% pairwise homology, up toabout 55% pairwise homology, up to about 60% pairwise homology, up toabout 65% pairwise homology, up to about 70% pairwise homology, up toabout 75% pairwise homology, up to about 80% pairwise homology, up toabout 85% pairwise homology, up to about 90% pairwise homology, up toabout 95% pairwise homology, up to about 96% pairwise homology, up toabout 97% pairwise homology, up to about 98% pairwise homology, up toabout 99% pairwise homology, up to about 99.5% pairwise homology, or upto about 99.9% pairwise homology with a second peptide. In some cases, apeptide of the disclosure can have at least about 20% pairwise homology,at least about 25% pairwise homology, at least about 30% pairwisehomology, at least about 35% pairwise homology, at least about 40%pairwise homology, at least about 45% pairwise homology, at least about50% pairwise homology, at least about 55% pairwise homology, at leastabout 60% pairwise homology, at least about 65% pairwise homology, atleast about 70% pairwise homology, at least about 75% pairwise homology,at least about 80% pairwise homology, at least about 85% pairwisehomology, at least about 90% pairwise homology, at least about 95%pairwise homology, at least about 96% pairwise homology, at least about97% pairwise homology, at least about 98% pairwise homology, at leastabout 99% pairwise homology, at least about 99.5% pairwise homology, atleast about 99.9% pairwise homology with a second peptide. Variousmethods and software programs can be used to determine the homologybetween two or more peptides, such as NCBI BLAST, Clustal W, MAFFT,Clustal Omega, AlignMe, Praline, or another suitable method oralgorithm.

In still other instances, the variant nucleic acid molecules of apeptide of any one of SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048 can be identified by either a determination of thesequence identity or homology of the encoded peptide amino acid sequencewith the amino acid sequence of any one of SEQ ID NO: 508—SEQ ID NO:758, SEQ ID NO: 798—SEQ ID NO: 1048, or by a nucleic acid hybridizationassay. Such peptide variants can include nucleic acid molecules (1) thatremain hybridized with a nucleic acid molecule having the nucleotidesequence of any one of SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048 (or any complement of the previous sequences) understringent washing conditions, in which the wash stringency is equivalentto 0.5×-2×SSC with 0.1% SDS at 55-65° C., and (2) that encode a peptidehaving at least 70%, at least 80%, at least 90%, at least 95% or greaterthan 95% sequence identity or homology to the amino acid sequence of anyone SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048.Alternatively, peptide variants of any one SEQ ID NO: 508—SEQ ID NO: 758or SEQ ID NO: 798—SEQ ID NO: 1048 can be characterized as nucleic acidmolecules (1) that remain hybridized with a nucleic acid molecule havingthe nucleotide sequence of any one SEQ ID NO: 508—SEQ ID NO: 758 or SEQID NO: 798—SEQ ID NO: 1048 (or any complement of the previous sequences)under highly stringent washing conditions, in which the wash stringencyis equivalent to 0.1×-0. 2×SSC with 0.1% SDS at 50-65° C., and (2) thatencode a peptide having at least 70%, at least 80%, at least 90%, atleast 95% or greater than 95% sequence identity or homology to the aminoacid sequence of any one of SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048.

Percent sequence identity or homology can be determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48:603(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1992). Briefly, two amino acid sequences are aligned to optimize thealignment scores using a gap opening penalty of 10, a gap extensionpenalty of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff(Id.). The sequence identity or homology is then calculated as: ([Totalnumber of identical matches]/[length of the longer sequence plus thenumber of gaps introduced into the longer sequence in order to align thetwo sequences])(100).

Additionally, there are many established algorithms available to aligntwo amino acid sequences. For example, the “FASTA” similarity searchalgorithm of Pearson and Lipman is a suitable protein alignment methodfor examining the level of sequence identity or homology shared by anamino acid sequence of a peptide disclosed herein and the amino acidsequence of a peptide variant. The FASTA algorithm is described byPearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and byPearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA firstcharacterizes sequence similarity by identifying regions shared by thequery sequence (e.g., SEQ ID NO: 485) and a test sequence that haseither the highest density of identities (if the ktup variable is 1) orpairs of identities (if ktup=2), without considering conservative aminoacid substitutions, insertions, or deletions. The ten regions with thehighest density of identities are then rescored by comparing thesimilarity of all paired amino acids using an amino acid substitutionmatrix, and the ends of the regions are “trimmed” to include only thoseresidues that contribute to the highest score. If there are severalregions with scores greater than the “cutoff” value (calculated by apredetermined formula based upon the length of the sequence and the ktupvalue), then the trimmed initial regions are examined to determinewhether the regions can be joined to form an approximate alignment withgaps. Finally, the highest scoring regions of the two amino acidsequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, Siam J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Illustrative parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity or homology ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from three to six, most preferably three, with otherparameters set as described above.

Some examples of common amino acids that are a “conservative amino acidsubstitution” are illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. The BLOSUM62 table is an aminoacid substitution matrix derived from about 2,000 local multiplealignments of protein sequence segments, representing highly conservedregions of more than 500 groups of related proteins (Henikoff andHenikoff, Proc. Nat? Acad. Sci. USA 89:10915 (1992)). Accordingly, theBLOSUM62 substitution frequencies can be used to define conservativeamino acid substitutions that may be introduced into the amino acidsequences of the present invention. Although it is possible to designamino acid substitutions based solely upon chemical properties (asdiscussed above), the language “conservative amino acid substitution”preferably refers to a substitution represented by a BLOSUM62 value ofgreater than −1. For example, an amino acid substitution is conservativeif the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or3. According to this system, preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 1 (e.g.,1, 2 or 3), while more preferred conservative amino acid substitutionsare characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Determination of amino acid residues that are within regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that can bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to, alignment of multiple sequences withhigh amino acid or nucleotide identity or homology and computer analysisusing available software (e.g., the Insight II. RTM. viewer and homologymodeling tools; MSI, San Diego, Calif), secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, G. J. , Current Opin. Struct. Biol. 5:372-6 (1995)and Cordes, M. H. et al., Current Opin. Struct. Biol. 6:3-10 (1996)). Ingeneral, when designing modifications to molecules or identifyingspecific fragments determination of structure can typically beaccompanied by evaluating activity of modified molecules.

Pairwise sequence alignment is used to identify regions of similaritythat may indicate functional, structural and/or evolutionaryrelationships between two biological sequences (protein or nucleicacid). By contrast, multiple sequence alignment (MSA) is the alignmentof three or more biological sequences. From the output of MSAapplications, homology can be inferred and the evolutionary relationshipbetween the sequences assessed. One of skill in the art would recognizeas used herein, “sequence homology” and “sequence identity” and “percent(%) sequence identity” and “percent (%) sequence homology” have beenused interchangeably to mean the sequence relatedness or variation, asappropriate, to a reference polynucleotide or amino acid sequence.

Chemical Modifications

A peptide can be chemically modified one or more of a variety of ways.In some embodiments, the peptide can be mutated to add function, deletefunction, or modify the in vivo behavior. One or more loops between thedisulfide linkages can be modified or replaced to include activeelements from other peptides (such as described in Moore and Cochran,Methods in Enzymology, 503, p. 223-251, 2012). Amino acids can also bemutated, such as to increase half-life or bioavailability, modify, addor delete binding behavior in vivo, add new targeting function, modifysurface charge and hydrophobicity, or allow conjugation sites.N-methylation is one example of methylation that can occur in a peptideof the disclosure. In some embodiments, the peptide can be modified bymethylation on free amines. For example, full methylation can beaccomplished through the use of reductive methylation with formaldehydeand sodium cyanoborohydride.

A chemical modification can, for instance, extend the terminalhalf-life, the absorption half-life, the distribution half-life of apeptide, change the biodistribution or pharmacokinetic profile, or themodification itself can be useful to provide viscosupplementation to ajoint. A chemical modification can comprise a polymer, a polyether,polyethylene glycol, a biopolymer, a polyamino acid, a fatty acid, adendrimer, an Fc region, a simple saturated carbon chain such aspalmitate or myristolate, sugars, hyaluronic acid, or albumin. Thechemical modification of a peptide with an Fc region can be a fusionFc-peptide. A polyamino acid can include, for example, a polyamino acidsequence with repeated single amino acids (e.g., polyglycine), and apolyamino acid sequence with mixed polyamino acid sequences (e.g.,gly-ala-gly-ala (SEQ ID NO: 1052)) that can or cannot follow a pattern,or any combination of the foregoing.

In some embodiments, the peptides of the present disclosure may bemodified such that the modification increases the stability and/or thehalf-life of the peptides. In some embodiments, the attachment of ahydrophobic moiety, such as to the N-terminus, the C-terminus, or aninternal amino acid, can be used to extend half-life of a peptide of thepresent disclosure. In other embodiments, the peptide of the presentdisclosure can include post-translational modifications (e.g.,methylation and/or amidation), which can affect, e.g., serum half-life.In some embodiments, simple carbon chains (e.g., by myristoylationand/or palmitylation) can be conjugated to the peptides. In someembodiments, for example, the simple carbon chains may render conjugatedpeptides easily separable from unconjugated material. For example,methods that may be used to separate the desired peptides of theinvention from unconjugated material include, but are not limited to,solvent extraction and reverse phase chromatography. In someembodiments, lipophilic moieties can be conjugated to the peptide andcan extend half-life through reversible binding to serum albumin.Moreover, the conjugated moieties can be lipophilic moieties that extendhalf-life of the peptides through reversible binding to serum albumin.In some embodiments, the lipophilic moiety can be cholesterol or acholesterol derivative including cholestenes, cholestanes,cholestadienes and oxysterols. In some embodiments, the peptides can beconjugated to myristic acid (tetradecanoic acid) or a derivativethereof. In other embodiments, the peptides of the present disclosureare coupled (e.g., conjugated) to a half-life modifying agent. Examplesof half-life modifying agents include but are not limited to: a polymer,a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, awater soluble polymer, a zwitterionic water soluble polymer, a watersoluble poly(amino acid), a water soluble polymer of proline, alanineand serine, a water soluble polymer containing glycine, glutamic acid,and serine, an Fc region, a fatty acid, palmitic acid, antibodies, or amolecule that binds to albumin.

In some embodiments, the first two N-terminal amino acids (GS) of SEQ IDNO: 485—SEQ ID NO: 758 can serve as a spacer or linker in order tofacilitate conjugation or fusion to another molecule, as well as tofacilitate cleavage of the peptide from such conjugated or fusedmolecules. In some embodiments, the peptides of the present disclosurecan be conjugated to other moieties that can modify or effect changes tothe properties of the peptides.

Active Agent Conjugates

Peptides according to the present disclosure can be conjugated or fusedto a peptide biological agent or other agent comprising amino acids(e.g., an antibody or antibody fragment, receptor or receptor fragment,ligand or ligand fragment, hormone or hormone fragment, growth factorsand growth factor fragments, biological toxins and fragments thereof, orother active portion of a peptide), a protein, a peptide, or to a smallmolecule, RNA, DNA, or other active agent molecular structure for use inthe treatment of cartilage diseases, disorders, or injuries. A peptideactive agent conjugate can be a peptide conjugated to an active agent byany mechanism described herein. For example, a peptide can be covalentlyconjugated to an active agent to form a peptide active agent conjugate.A peptide can be chemically conjugated to an active agent to form apeptide active agent conjugate. A peptide and active agent can beexpressed as a fusion protein to form a peptide active agent conjugate.For example, an antibody or fragment thereof and a peptide can beexpressed as a fusion protein to form a peptide active agent conjugate.For example, in certain embodiments, a peptide as described herein canbe fused to another molecule, such as an active agent that provides afunctional capability. A peptide can be conjugated with an active agentthrough expression of a vector containing the sequence of the peptidewith the sequence of the active agent. In various embodiments, thesequence of the peptide and the sequence of the active agent areexpressed from the same Open Reading Frame (ORF). In variousembodiments, the sequence of the peptide and the sequence of the activeagent can comprise a contiguous sequence. Various vectors andrecombinant systems known in the art can be employed to make such fusionpeptides. The peptide and the active agent can each retain similarfunctional capabilities in the fusion peptide compared with theirfunctional capabilities when expressed separately.

Furthermore, for example, in certain embodiments, the peptides describedherein are attached to another molecule, such as an active agent thatprovides a functional capability. In some embodiments, 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 active agents can be linked to a peptide. Multiple activeagents can be attached by methods such as conjugating to multiple lysineresidues and/or the N-terminus, or by linking the multiple active agentsto a scaffold, such as a polymer or dendrimer and then attaching thatagent-scaffold to the peptide (such as described in Yurkovetskiy, A. V., Cancer Res 75(16): 3365-72 (2015).

Described herein are active agents that can be conjugated to thepeptides of the present invention for use in either cartilage disordersor kidney disorders, or both. In some embodiments, certain compounds ordrugs are appropriate for use in either cartilage or kidney disorders,certain drug classes may be preferred for specific treatment dependingon the indication or disorder. As described herein, it is understoodthat certain active agents are described in a non-limiting exemplarymanner for use in treatments of cartilage and/or kidney indications. Oneor more of such active agents can be conjugated to a peptide of thepresent invention alone or in combination with one or more detectableagents described herein. In some embodiments, active agents that can beconjugated to any peptide of this disclosure can be classified bymechanism. For example, active agents can belong to the class ofanti-inflammatory drugs, immunosuppressive (immune suppression) drugs,analgesics/pain relief drugs, disease modifying osteoarthritic drugs(DMOADs), cell depleting agents/apoptosis modifiers, bone resorptiveagents and viscosupplementing agents, and tissue normalization (diseasemodifying) drugs.

Anti-inflammatory active agents can include, but are not limited to,corticosteroids, glucocorticoids, nonsteroidal anti-inflammatory drugs(NSAIDs), biologics, and other small molecules. Examples ofcorticosteroid active agents that can be conjugated to any peptide ofthis disclosure for delivery to the joints and kidneys includetriamcinolone dexamethasone, budesonide, and triamcinolone acetonide.Examples of NSAID active agents that can be conjugated to any peptide ofthis disclosure for delivery to the joints and kidneys include naproxenand ibuprofen. Other active agents include acetylsalicylic acid andacetaminophen. NSAID active agents can be further classified into COX²inhibitors. An example of a COX² inhibitor active agent directed to aprostaglandin pathway that can be conjugated to any peptide of thisdisclosure for delivery to the joint includes celecoxib. An example of aCOX² inhibitor active agent with anti-leukotriene receptor antagonistthat can be conjugated to any peptide of this disclosure for delivery tothe joint includes montelukast. An example of a COX² inhibitor activeagent that can be conjugated to any peptide of this disclosure fordelivery to the kidneys includes iguratimod. Biologic active agents canbe further classified into active agents that are IL-1 familyinhibitors, IL-17 or IL-23 pathway inhibitors, IL-6 family inhibitors,interferon receptor inhibitors, tumor necrosis factor (TNF) inhibitors,RANK pathway inhibitors, B cell inhibitors, anti-IgE active agents, andco-stimulation inhibitors. An example of an IL-1 family inhibitor activeagent that can be conjugated to any peptide of this disclosure fordelivery to the joints includes anakinra An example of an IL-17/IL-23pathway inhibitor active agent that can be conjugated to any peptide ofthis disclosure for delivery to the joints includes secukinumab. Anexample of an IL-6 family inhibitor active agent that can be conjugatedto any peptide of this disclosure for delivery to the kidneys includessirukumab. An example of an interferon receptor inhibitor active agentthat can be conjugated to any peptide of this disclosure for delivery tothe kidneys includes anifrolumab. An example of a TNF inhibitor activeagent that can be conjugated to any peptide of this disclosure fordelivery to the joints includes infliximab or etanercept. An example ofa RANK pathway inhibitor active agent that can be conjugated to anypeptide of this disclosure for delivery to the joints includesdenosumab. An example of a B cell inhibitor active agent that can beconjugated to any peptide of this disclosure for delivery to the jointsand kidneys includes rituximab. An example of an anti-IgE active agentthat can be conjugated to any peptide of this disclosure for delivery tothe kidneys includes omalizumab. An example of a co-stimulationinhibitor active agent that can be conjugated to any peptide of thisdisclosure for delivery to the joints includes abatacept.

Pain relief active agents can include, but are not limited toanalgesics, counter-irritants, and pain receptor blocking drugs.Analgesics can be further classified into non-narcotic agents andnarcotic agents. An example of a non-narcotic active agent that can beconjugated to any peptide of this disclosure for delivery to the jointsincludes acetaminophen. An example of a narcotic active agent that canbe conjugated to any peptide of this disclosure for delivery to jointsincludes oxycodone. Counter-irritant active agents can be furtherclassified as natural products. An example of a natural product that canbe conjugated to any peptide of this disclosure for delivery to thejoints includes capsaicin. Pain receptor blocking active agents can befurther classified as TRPV4 inhibitors. An example of a TRPV4 inhibitoractive agent that can be conjugated to any peptide of this disclosurefor delivery to the joints includes GSK2193874.

Apoptosis modifier active agents can include, but are not limited to,biologics and small molecules. Biologic apoptosis modifier active agentscan be further classified as Fas/FasL inhibitors, TNF/TNFR inhibitors,TRAIL/TRAILR inhibitors, TWEAK/Fn14 inhibitors, IL-1 inhibitors, IL-1receptor antagonists, growth factors, and sclerostin inhibitors. Anexample of a TNF/TNFR inhibitor active agent that can be conjugated toany peptide of this disclosure for delivery to the joints includesinfliximab. An example of a TRAIL/TRAILR inhibitor active agent that canbe conjugated to any peptide of this disclosure for delivery to thejoints includes osteoprotegrin. An example of a TWEAK/Fn14 inhibitoractive agent that can be conjugated to any peptide of this disclosurefor delivery to the kidneys includes BIIB023. An example of an IL-1receptor antagonist that can be conjugated to any peptide of thisdisclosure for delivery to the joints includes anakinra An example of agrowth factor active agent that can be conjugated to any peptide of thisdisclosure for delivery to the joints includes IGF-1. An example of agrowth factor active agent that can be conjugated to any peptide of thisdisclosure for delivery to the kidneys includes EGF. An example of asclerostin inhibitor active agent that can be conjugated to any peptideof this disclosure for delivery to the joints includes romosozumab.Small molecule apoptosis modifier active agents can be furtherclassified as caspase inhibitors, iNOS inhibitors, surfactants, andbisphosphonates. An example of a caspase inhibitor active agent that canbe conjugated to any peptide of this disclosure for delivery to thejoints includes ZVAD-fmk. An example of an iNOS inhibitor active agentthat can be conjugated to any peptide of this disclosure for delivery tothe joints include S-methylisothiourea. An example of a surfactantactive agent that can be conjugated to any peptide of this disclosurefor delivery to the joints include P188. An example of a bisphosphonateactive agent that can be conjugated to any peptide of this disclosurefor delivery to the joints includes alendronate. Moreover, the knownclass of drugs called senotherapeutics, also referred to as senolyticsor senolytic drugs or senolytic compounds, refers to small moleculesthat can selectively induce death of senescent cells and for example bydirectly or indirectly inducing apoptosis in senescent cells. Inaddition, senolytics may also act via non-apoptotic mechanisms of celldeath including by necroptis, autophagic cell death, pyroptis andcaspase-independent cell death (Journal of Cell Science 127; 2135-2144(2014)). Such drugs can attenuate age-related deterioration of tissuesor organs. Examples of drugs that can be conjugated to any peptide ofthis disclosure to induce apoptosis or induce cell death vianon-apoptotic mechanisms include quercetin, dasatinib, bortezomib,carfilzomib, and navitoclax amongst other compounds disclosed herein.Additional active agents are described in the following references: Zhu,Y et al., Aging Cell 14(4):644-58 (2015); Kirkland, J L, Exp Gerontol.48(1): 1-5 (2013); Kirkland J L and Tchkonia T, Exp Gereontol. 68: 19-25(2015) Tchkonia, T et al., J Clin Invest., 123(3): 966-72 (2013);WO2016118859; Sugumar, D et al., Pharmagenomics Pers Med. 8: 23-33(2015); Jiafa, R et al., Sci Rep. 6: 23968 (2016); Swanson, CD et al.,Nat Rev Rheumatol., 5(6): 317-324 (2009); Oh, CJ et al., PLoS One, 7(10):e45870 (2012); and Adebajo, A and Boehncke, R^(a), Psoriatic Arthritisand Psoriasis: Pathology and Clinical Aspects, Springer (2016).

Tissue normalization (disease modifying) active agents can include, butare not limited to, biologics and small molecules. Biologic activeagents can be further classified as chemokines (e.g., for stem cellrecruitment) and growth factors. An example of a tissue normalizationchemokine active agent that can be conjugated to any peptide of thisdisclosure for delivery to the joints includes MIP-3α. An example of atissue normalization growth factor active agent that can be conjugatedto any peptide of this disclosure for delivery to the joints includesBMP-2. Small molecule active agents can be further classified asflavonoids, ACE inhibitors, and anti-proliferative active agents. Anexample of a tissue normalization flavonoid active agent that can beconjugated to any peptide of this disclosure for delivery to the jointsincludes icariin. An example of a tissue normalization ACE inhibitoractive agent that can be conjugated to any peptide of this disclosurefor delivery to the kidneys includes captopril. An example of a tissuenormalization anti-proliferative active agent that can be conjugated toany peptide of this disclosure for delivery to the joints includesmethotrexate.

TABLE 53 describes active agents for treatment of a cartilage disorderthat can be conjugated to any peptide of the present disclosure to formpeptide-drug conjugates.

TABLE 53 Exemplary Active Agents for Cartilage Disorders Active AgentClass Active Agent Gold compound Gold Gold compound Auranofin Goldcompound Gold Sodium Thiomalate Gold compound Gold Thioglucose Goldcompound Thiomalic Acid Gold compound Gold Thiosulphate AnalgesicsTramadol (e.g., Ultram, Ultracet) and derivatives Analgesics Oxycodone(e.g., Percocet, Oxycontin) and derivatives Analgesics Hydrocodone(e.g., Norco, Vicoprofen) Analgesics Morphine Analgesics FentanylAnalgesics Oxymorphone Analgesics Hydromorphone Analgesics MeperidineAnalgesics Buprenorphine Analgesics Methadone Bisphosphonate AlendronateBisphosphonate Ibandronate Bisphosphonate Risedronate BisphosphonatePamidronate Bisphosphonate Zoledronate Non-Nitrogen Containing FirstClodronate Generation Bisphosphonate Non-Nitrogen Containing FirstEtidronate Generation Bisphosphonate Non-Nitrogen Containing FirstTiludronate Generation Bisphosphonate Apoptosis InhibitorsOsteoprotegerin (OPG) Sclerostin Antagonist Apoptosis AMG785(Romosozumab) Inhibitors Caspase-1 ICE Inhibitors VX-740 (Pralnacasan)Counter-irritants Menthol Counter-irritants Capsaicin RANKL TargetingAgents Denosumab Cathepsin K Targeting Agents Odanacatib TNF-αAntagonists CDP571 TNF-α Antagonists ISIS 104838 Anti-Pain DrugsDuloxetine Polymers Low Molecular Weight Chitosan Matrix DrugsChondroitin sulfate glucosamine Cytokines/Growth Factors TGF-beta MatrixLaminin Matrix Fibronectin Matrix Lubricin Matrix Hyaluronic acidinjections Matrix Glucosamine Immunosuppressants Rapamycin HIF-1αModulators HIF-2α Modulators Corticosteroid Tixocortol pivalateGlucocorticoid Corticosteroid Hydrocortisone Acetate GlucocorticoidCorticosteroid Hydrocortisone t-Butyl Acetate GlucocorticoidCorticosteroid Prednisolone Acetate Glucocorticoid CorticosteroidPrednisolone t-Butyl Acetate Corticosteroid Dexamethasone AcetateCorticosteroid Dexamethasone t-Butyl Acetate GlucocorticoidCorticosteroid Triamcinolone Diacetate

TABLE 54 describes active agents for treatment of a kidney disorder thatcan be conjugated to any peptide of the present disclosure to formpeptide-drug conjugates.

TABLE 54 Exemplary Active Agents for Kidney Disorders Active Agent ClassActive Agent ACE Inhibitors Captopril Angiotensin receptor blockersAngiotensin receptor blocker losartan (Cozaar) HormonesAdrenocorticotropic hormone Hormones corticotropin-releasing hormoneamphotericin B digitalis glycosides potassium-depleting diureticsCoumarine anticoagulants NLRP3 Inflammosome Targeted MCC950 Drugs NLRP3Inflammosome Targeted BHB Drugs NLRP3 Inflammosome Targeted Type Iinterferon Drugs NLRP3 Inflammosome Targeted IFN-beta Drugs NLRP3Inflammosome Targeted Resveratrol Drugs NLRP3 Inflammosome TargetedArglabin Drugs NLRP3 Inflammosome Targeted CB2R agonist Drugs NLRP3Inflammosome Targeted MicroRNA-223 Drugs

TABLE 55 describes active agents for treatment of a cartilage disorderand a kidney disorder that can be conjugated to any peptide of thepresent disclosure to form peptide-drug conjugates.

TABLE 55 Exemplary Active Agents for Cartilage Disorders and KidneyDisorders Active Agent Class Active Agent IL-6 Receptor ModulatorsTocilizumab IL-6 Receptor Modulators Sarilumab IL-6 Receptor ModulatorsALX-0061 IL-6 Receptor Modulators Sirukumab IL-6 Receptor ModulatorsClazakizumab IL-6 Receptor Modulators Olokizumab IL-6 ReceptorModulators MEDI5117 IL-17 Antagonists Secukinumab IL-17 AntagonistsBrodalumab IL-17 Antagonists Ixekizumab Antagonists of p40 Subunit ofIL- Ustekinumab 12/IL-23 Antagonists of p40 Subunit of IL- Briakinumab12/IL-23 Antagonists of p19 Subunit of IL-23 Tildrakizumab Antagonistsof p19 Subunit of IL-23 Guselkumab IL-23 Antagonists Soluble IL-23 (orcytokine-binding homology region of soluble IL-23) IL-1 AntagonistsCanakinumab IL-1 Antagonists Rilonacept IL-1 Antagonists GevokizumabIL-1 Antagonists LY2189102 IL-1 Antagonists Lentiviral-mediated RNAiIL-12 Antagonists IL-1 Receptor Antagonists Anakinra IL-1 ReceptorAntagonists MEDI-8968 IL-1 Receptor Antagonists AMG-108 IL-1 ReceptorKineret Interleukins/Pro-Inflammatory Pro-inflammatory IL-1α or IL-1βCytokines Interleukins IL-8 Interleukins IL-15 Interleukins IL-18Interleukins IL-4 Interleukins IL-10 Interleukins IL-13 InterleukinsIL-22 Interleukins IL-17 p38 Inhibitors VX-745 p38 Inhibitors BIRB 796p38 Inhibitors SCIO-469 p38 Inhibitors VX-702 p38 Inhibitors Pamapimodp38 Inhibitors ARRY-797 Corticosteroids 17-monopropionateCorticosteroids Desciclesonide Corticosteroids FlunisolideCorticosteroids Mometasone furoate Corticosteroids 22-hydroxyintermediate budesonide derivative Corticosteroids 6β-hydroxy budesonidederivative Corticosteroids Δ6-budesonide derivative Corticosteroids23-hydroxy budesonide derivative Corticosteroids16α-butryloxyprednisolone budesonide derivative Corticosteroids16α-hydroxyprednisolone budesonide derivative Corticosteroid(Beclomethasone) QVAR inhalation Corticosteroid (Budesonide) pulmicortrespules Corticosteroid Flovent HFA 44 Corticosteroid (Mometasone)Asmanex HFA Corticosteroid (Mometasone) Budesonide symbicortCorticosteroid Dexamethasone sodium phosphate Corticosteroid Tixocortolpivalate Corticosteroid Ciclesonide Glucocorticoids 21-nortriamcincoloneacetonide Glucocorticoids Δ6-triamcinolone Glucocorticoids 6b-hydroxytriamcinolone acetonide Glucocorticoids 21-carboxy triamcinoloneacetonide Glucocorticoids 6b-OH, 21-COOH triamcinolone acetonideGlucocorticoids 6α fluorocortisol Glucocorticoids 9α fluorocortisolGlucocorticoids Δ1-dehydro configuration in prednisolone Glucocorticoids16-methylene dexamethasone derivative Glucocorticoids 16α-methyldexamethasone derivative Glucocorticoids 16β-methyl betamethasonederivative Glucocorticoids Cyclophosphamide GlucocorticoidsMycophenolate Glucocorticoids/Mineralocorticoids CortisolGlucocorticoids/Mineralocorticoids HydrocortisoneGlucocorticoids/Mineralocorticoids PrednisoloneGlucocorticoids/Mineralocorticoids Betamethasone GlucocorticoidFluticasone Glucocorticoid Fluticasone propionate Steroid (flunisolide)Aerobid Steroid (flunisolide) Aerobid-M Steroid (flunisolide) AerospanSteroid (Flunisolide) Fluticasone Furoate Steroid (Fluticasone) FloventHFA 110 Steroid (Fluticasone) Flovent HFA 220 Steroid (Fluticasone)Flovent Diskus 50 Steroid (Fluticasone) Asmanex Steroid Betamethasoneacetate Steroid Betamethasone sodium phosphate Steroid Betamethasonevalerate Steroid Beclomethasone dipropionate Local Anesthetic procainehydrochloride Local Anesthetic Novacain Anesthetic bupivacainehydrochloride Anesthetic lidocaine hydrochloride Local Anestheticropivacaine hydrochloride Analgesics Morphine Analgesics FentanylQuinazolines Feitinib/Iressa Quinazolines Sorafenib/Nexavar QuinazolinesLapatinib ditosylate/Tykerb/Tyverb Quinazolines Sunitinib/SutentQuinazolines Bortezomib/Velcade/Cytomib QuinazolinesEverolimus/Temsirolimus Quinazolines Inhibitors of IAPS QuinazolinesActivators of caspase pathway Quinazolines Activators of AKT pathwayQuinazolines Propylpeptidase inhibitors Quinazolines Activators of p53Quinazolines Inhibitors of anti-apoptotic protein inhibitors ProlylHydroxylase (PHD) Desferrioxamine Inhibitors Prolyl Hydroxylase (PHD)Dimethyloxalylglycine (DMOG) Inhibitors Prolyl Hydroxylase (PHD)L-mimosine (L-mim) Inhibitors Aptamers Peptide aptamers Aptamers RNAaptamer A-p50 Aptamers Peptide A aptamer TrxLef1D Aptamers Aptamer E07Aptamers Aptamer gemcitabine polymers Aptamers RAGE Aptamers PegaptanibProteosome Inhibitors Bortezomib Proteosome Inhibitors CarfilzomibSecond Generation Proteosome Ixazomib Inhibitors Second GenerationProteosome Delanzomib Inhibitors Second Generation Proteosome OprozomibInhibitors Second Generation Proteosome Marizomib Inhibitors ApoptosisInhibitors FLIP agonist Apoptosis Inhibitors nitric oxide synthaseinhibitors Apoptosis Inhibitors caspase-3 inhibitors (Z-DEVD-fmk (SEQ IDNO: 1053)) Apoptosis Inhibitors caspase-9 inhibitors (Z-LEHD-fmk (SEQ IDNO: 1054)) Apoptosis Inhibitors Sclerostin antagonists ApoptosisInhibitors/Growth Factor IGF-1 BCL-2 Agonist Apoptosis InhibitorsOblimersen BCL-2 Agonist Apoptosis Inhibitors Obatoclax BCL-2 AgonistApoptosis Inhibitors Navitoclax BCL-2 Agonist Apoptosis InhibitorsVenetoclax (ABT-199) BCL-2 Agonist Apoptosis Inhibitors Navotoclax(ABT-263) BCL-2 Agonist Apoptosis Inhibitors GX01 series of compoundsBCL-2 Agonist Apoptosis Inhibitors BCL-2 small molecule antagonistsBCL-2 Agonist Apoptosis Inhibitors Tetraocarcin-A derivatives BCL-2Agonist Apoptosis Inhibitors Chelerythrine BCL-2 Agonist ApoptosisInhibitors Antimycin A derivatives BCL-2 Agonist Apoptosis InhibitorsHA14-1 BCL-2 Agonist Apoptosis Inhibitors Synthetic compound antagonistof BH3 BCL-2 Agonist Apoptosis Inhibitors Genasense BCL-2 AgonistApoptosis Inhibitors ISIS 22783 BCL-2/BCL-XL Agonist ApoptosisBispecific Antisense Inhibitors Proapoptotic BCL-2 Targeting Bax, Bak,Bid, Bad-derived BH3 Peptides Drugs Proapoptotic BCL-2 Targeting SAHBsDrugs Proapoptotic BCL-2 Targeting BH3Is Drugs BCL-2/BCL-XL AgonistApoptosis ABT-737 Inhibitors BCL-X Inhibitors Apoptosis ModifiersCaspase-1 Inhibitors Apoptosis Modifiers Caspase-8 InhibitorsPan-caspase Caspase Inhibitor IDN-6556 Pan-caspase Caspase InhibitorIDN-6734 Pan-caspase Caspase Inhibitor VX-799 Pan-caspase InhibitorMX1013 Pan-caspase Caspase Inhibitor M-920 Pan-caspase Caspase ActivatorMX-2060 derivatives Pan-caspase Caspase Activators Small-moleculecompounds Pan-caspase Caspase Activators RGD peptides Pan-caspaseinhibitors ZVAD-fmk Caspase-1 ICE Inhibitors IDN-11104 Caspase-1 ICEInhibitors VX-756 Caspase-3 Inhibitors M-826 Caspase-3 Inhibitors M-791Caspase-3 Inhibitors Immunocasp-3 Caspase-3 Inhibitors Ad-G/iCasp3Caspase-3 Inhibitors PEF-F8-CP3 Caspase-6 Inhibitors Immunocasp-6Caspase-9 Inhibitors FKBP12/caspase-9 fusion protein IAP AntagonistsBIR3 antagonists XIAP Antagonists Capped tripeptide XIAP AntagonistsXIAP Antagonists Smac-mimetic compounds XIAP AntagonistsAEG35156/GEM ®640 XIAP Inhibitors Embelin XIAP Inhibitors XIAP antisenseand RNA constructs XIAP/cIAP-1/cIAP-2 Inhibitors Small molecule SMACmimetics IAP/Caspase Inhibitors HIV-Tat/polyarginine-conjugated SMACpeptides BIR2/Caspase-3 Inhibitors TWX024 BIR2 Inhibitors Polyphenylureaderivatives Survivin Targeting Drugs LY2181308 Survivin Targeting DrugsAd-Survivin T34A Anti-TWEAK Apoptosis Modifiers BIIB023 Xanthine OxidaseInhibitors Allopurinol Xanthine Oxidase Inhibitors Febuxostat XanthineOxidase Inhibitors Zyloprin Growth Factor bFGF Growth Factor IGF GrowthFactor TFG-beta Growth Factor BMP-2 Growth Factor BMP-9 Growth FactorBMP-13 Growth Factor BMP-7 Growth Factor BMP-3 inhibitors Growth FactorTFG-β1 Growth Factor OP-1 Growth Factor PDGF Growth Factor PTH GrowthFactor PTHrP Growth Factor MIP-3α Growth Factor EPO Growth Factor FGFGrowth Factor FGF-2 Growth Factor FGF-18 Growth Factor TGF-β3 GrowthFactor VEGF Growth Factor Wnt proteins Growth Factor EGF Growth FactorGM-CSF Flavonoid Icariin Flavonoid Quercetin Tyrosine Kinase Inhibitor(Lck/Btk Dasatinib Inhibitor) TRPV4 Activators GSK1016790A TRPV4Activators 4alpha-PDD TRPV4 Inhibitors HC-067047 TRPV4 InhibitorsGSK2193874 NSAID Ampion NSAID Phenylbutazone NSAID Naproxen lysozymeconjugate NSAID Acetal salicylic acid DMARDs Sulfasalazine DMARDsLeflunomide DMARDs Hydroxychloroquine (Plaquenil) Disease-ModifyingOsteoarthritis FGF-18 Drugs (DMOADs) Uricosurics Sulfinpyrazone MSCMatrix Collagen MSC Matrix Fibrin MSC Matrix Polylactatous SurfactantP188 and other surfactants Molecules for Bone Marrow Niches AngiopoetinMolecules for Bone Marrow Niches Bone morphogenitic proteins Moleculesfor Bone Marrow Niches Epinephrine Molecules for Bone Marrow NichesNorepinephrine Molecules for Bone Marrow Niches GDF5 Molecules for BoneMarrow Niches ICAN1 Molecules for Bone Marrow Niches Jagged1 Moleculesfor Bone Marrow Niches Osteopontin Molecules for Bone Marrow Nichesparathyoid hormone Molecules for Bone Marrow Niches Calcitonin Moleculesfor Bone Marrow Niches steel factor Molecules for Bone Marrow NichesThrombopoetin Molecules for Bone Marrow Niches vascular cell adhesionmolecule 1 Chemokine Molecules for Bone CXCL12 Marrow Niches B CellTargeting Agents Rituximab B Cell Targeting Agents BLys B Cell TargetingAgents TACI T Cell Co-stimulation Antagonists Abatacept JAK TargetingAgents Tofacitinib Calcineurin Inhibitors Tacrolimus CalcineurinInhibitors Cyclosporin Calcineurin Inhibitors Voclosporin COX-2Inhibitors Iguratimod COX-2 Inhibitors Montelukast COX-2 InhibitorsRofecoxib COX-2 Inhibitors Valdecoxib Interferon Receptor InhibitorsAnifrolumab IFN-α Inhibitors Sifalimumab Anti-IgE Agents Omalizumab iNOSInhibitors S-methylisothiourea CD20 Antagonists/B Cell InhibitorsOcrelizumab BAFF Antagonists/B Cell Inhibitors Belimumab TNF SuperfamilyBAFF and APRIL Atacicept Antagonists/B cell Inhibitors TNF-α AntagonistsThalidomide TNF-α Antagonists Lenalidomide TNF-α AntagonistsPomalidomide TNF-α Antagonists Pentocifylline TNF-α AntagonistsBupropion TNF Antagonists Lentiviral-mediated RNAi TNF AgonistsRecombinant TNF-α TRAIL Receptor Agonists HGS-ETR1 TRAIL ReceptorAgonists HGS-ETR2 TRAIL Receptor Agonists HGS-TR2J TRAIL ReceptorAgonists PRO1762 TRAIL Receptor Agonists TRA-8 CD95/Fas Agonists CD95-FcMarine Bioactive Compounds TRAIL-Resistance Overcoming Marine BioactiveCompounds Marine Bioactive Compounds mazamine A Marine BioactiveCompounds marine-derived chroomycins Marine Bioactive CompoundsCarotenoids Marine Bioactive Compounds Aplysin Marine BioactiveCompounds Aplidin Marine Bioactive Compounds Siphonaxanthin MarineBioactive Compounds pectinotoxin-2 Anti-Complement Drugs EculizumabPAR-2 Modulators Pepducin P2pal-18 miR-2013 Blockers Anti-senseoligonucleotides Nrf2 Activator Dimethyl fumarate p53 Targeting DrugsINGN201 p53 Targeting Drugs SCH58500 p53 Targeting Drugs ONYX-015 p53Targeting Drugs C-terminal p53 peptides p53 Targeting Drugs CDB3 p53Targeting Drugs CP31398 p53 Targeting Drugs Prima-1 p53 Targeting DrugsHPV E6-binding peptide aptamers p53 Targeting Drugs Nutlins p53Targeting Drugs Chalcones p53 Targeting Drugs Small peptides p53Targeting Drugs Pifithrin-α p53 Targeting Drugs/Apoptosis QP1-1002Modifiers (T cells) Apaf-1 Targeting Drugs/Apoptosis QM56 Modifiers (Tcells) Apaf-1 Targeting Drugs/Apoptosis SVT016426 Modifiers (T cells)Ferrostatin 16/86 BASP1 Targeting Drugs/Apoptosis BASP siRNA Modifiers(T cells) Anti-Inflammatory Drugs CCX140 Anti-Inflammatory Drugs CXA-10Anti-Inflammatory Drugs/Anti- Alkaline phosphatase Fibrotic DrugsAnti-Fibrotic Drugs Dnmt1 inhibitors Anti-Inflammatory Drugs/ApoptosisTHR-184 Modifiers (T cells) Immunosuppressants Lithium β2-AdrenergicAgonists Formoterol Anti-Inflammatory Drugs CRMD-001 Endothelin-1Targeting Drugs Astrasentan Vasopressin Receptor Antagonists TolvaptanVasopressin Receptor Antagonists RWJ-676070 ImmunosuppressantsAzathioprine Immunosuppressants Mycophenolic acid ImmunosuppressantsCyclosporine Immune Modulators Laquinimod Slow-acting antirheumaticdrugs (SAARDs) Colcrys Hormones parathyroid hormone Hormones growthhormone 11-beta hydroxysteroid dehydrogenases MineralocorticoidProopiomelanocortin fludrocortisonesoxycorticosterone acetate vaccinesfrom live attenuated viruses Aspirin Insulin Isonizaid Oral hypoglycemicagents Antacids Carbamazepine Cholestyramine Colestipol EphedrineErythromycin Mitotane oral contraceptives Phenobarbital PhenytoinRifampin Troleandomycin Non-selective caspase inhibitor okadaic acidCamptothetic Staurosporine HFA Alvesco inhalation Breo Ellipta AdvairMometasone Dulera Umeclidinium Anoro Reactive Oxygen Species TargetingDrugs Cytokines/Growth Factors TGF-beta NOD-like receptor protein 3-dependent caspase 1 Targeting Drugs NSAID Etoricoxib Apoptosis ModifiersMCL1 inhibitors Teriparatide BH3 mimetics AZD 4320 Carrier Proteins Lowmolecular weight human serum albumin Ceramide Targeting Drugs DMARDsPenicillamine Chondrogenic factors Anti-oxidative factors A(1)AR agonistS1P(2)R antagonist Antimalarials BAX/BAK activating drugs Selective GRActivators (SEGRAs) Rap1 Targeted Drugs Senolytic Ephrin Ligand (EFN) B1blockers Senolytic Cyclin-dependent kinase inhibitor 1A (p21)phosphatidylinositol-4,5-bishophate 3-kinase delta catlyatic subunit(PI3KCD) blockers Senolytic Plasminogen-activated inhibitor-2 (PAI-2)blockers Senesce-associated secretory phenotype (SASP) inhibitorsHormone Tetracosactide

TABLE 56 describes additional active agents for treatment of a cartilagedisorder and a kidney disorder that can be conjugated to any peptide ofthe present disclosure to form peptide-drug conjugates.

TABLE 56 Exemplary Active Agents for Cartilage Disorders and KidneyDisorders Active Agent Class Active Agent Peptide Oligopeptide PeptidePolypeptide Peptide Peptidomimetic Nucleic Acid Polynucleotide NucleicAcid Polyribonucleotide Nucleic Acid Oligonucleotide Nucleic Acid DNANucleic Acid cDNA Nucleic Acid ssDNA Nucleic Acid RNA Nucleic Acid dsRNANucleic Acid micro RNA Nucleic Acid Interfering RNA Nucleic Acid AptamerAntibody single chain variable Fragment (scFv) Antibody AntibodyFragment Antibody Aptamer Antibody Fc domains Antibody Fc regionsAntibody Fc active fragments or modifications thereof Cytokine Cytokineantagonists Mavrilimumab Cytokine antagonists Ixekizumab Cytokineantagonists Tocilizumab Cytokine antagonists Anakinra Cytokineantagonists Ustekinumab Cytokine antagonists Secukinumab InterferonHormone Enzymes Growth Factor Checkpoint Inhibitor CD Antigen ChemokinesNeurotransmitters Ion Channel Inhibitors G-protein coupled receptorinhibitors G-protein coupled receptor activators Tumor necrosis factorinhibitors Chemical Agents Radiosensitizers RadioprotectantsRadionuclide Therapeutic Small Molecules Steroids CorticosteroidsAnti-inflammatory Agents Immune Modulators Abatacept Immune ModulatorsRituximab Complement Fixing Peptides or Proteins Tumor Necrosis FactorFamily Tumor Necrosis Factor (TNF) soluble receptor or antibodyInhibitors Tumor Necrosis Factor Family Activators Tumor Necrosis Factor(TNF) soluble receptor or antibody Caspase protease inhibitors oractivators NF-kB, RIPK1 and/or RIPK3 Inhibitors NF-kB, RIPK1 and/orRIPK3 Activators Death-receptor ligand activator or inhibitor TumorNecrosis Factor Family TNFR1 Agonists Tumor Necrosis Factor Family TNFR2Agonists Tumor Necrosis Factor Family CD27/TNFRSF7 Agonists TumorNecrosis Factor Family CD30/TNFRSF8 Agonists Tumor Necrosis FactorFamily OX40/TNFRSF4 Agonists Tumor Necrosis Factor Family CD40/TNFRSF5Agonists Tumor Necrosis Factor Family 4-1BB/TNFRSF9 Agonists TumorNecrosis Factor Family RANK (receptor activator of NF-kappa B/TNFRSF11A)Agonists Tumor Necrosis Factor Family TWEAK receptor/TNFRSF12A AgonistsTumor Necrosis Factor Family TAC1/TNFRSF13B Agonists Tumor NecrosisFactor Family BAFF-R (BAFF receptor/TNFRSF13C) Agonists Tumor NecrosisFactor Family HVEM (herpes virus entry mediator/TNFRSF14) Agonists TumorNecrosis Factor Family RELT/TNFRSF19L Agonists Tumor Necrosis FactorFamily ectodysplasin A2 isoform receptor/TNFRS27 Agonists Tumor NecrosisFactor Family ectodysplasin A1 Agonists TNF Family Member AnhidroticReceptor Tumor Necrosis Factor Family Decoy Receptor 3/TNFRSF6BAntagonists Tumor Necrosis Factor Family Decoy Receptor 1/TNFRSF10CAntagonists Tumor Necrosis Factor Family Decoy Receptor 2/TNFRSF10DAntagonists Tumor Necrosis Factor Family DR3 (death receptor 3/TNFRSF25)Antagonists Tumor Necrosis Factor Family DR4 (death receptor4/TNFRSF10A) Antagonists Tumor Necrosis Factor Family DR5 (deathreceptor 5/TNFRSF10B) Antagonists Tumor Necrosis Factor Family DR6(death receptor 6/TNFRSF21) Antagonists Tumor Necrosis Factor FamilyFas/TNFRSF6 Antagonists Tumor Necrosis Factor Family Lymphotoxin breceptor/TNFRS3 Antagonists Tumor Necrosis Factor Family OPG(osteoprotegerin/TNFRSF11B) Antagonists Tumor Necrosis Factor FamilyNerve Growth Factor Receptor/TNFRSF16 Antagonists Tumor Necrosis FactorFamily BCMA (B Cell Maturation Antigen/TNFRSF17) Antagonists TumorNecrosis Factor Family GITR (Glucocorticoid-Induced TNFReceptor/TNFRSF18) Antagonists Tumor Necrosis Factor Family TAJ(Toxicity and JNK Inducer/TNFRSF19) Antagonists Tumor Necrosis FactorFamily TNFRSF22 Antagonists Tumor Necrosis Factor Family TNFRSF23Antagonists TNF Receptor Superfamily Ligands TNF alpha TNF ReceptorSuperfamily Ligands Lymphotoxin-a TNF Receptor Superfamily Ligands TumorNecrosis Factor Membrane Form TNF Receptor Superfamily Ligands TumorNecrosis Factor Shed Form TNF Receptor Superfamily Ligands LIGHT TNFReceptor Superfamily Ligands Lymphotoxin b2a1 heterotrimer TNF ReceptorSuperfamily Ligands OX-40 Ligand TNF Receptor Superfamily LigandsCompound 1 [PMID: 24930776] TNF Receptor Superfamily Ligands CD40 LigandTNF Receptor Superfamily Ligands Fas Ligand TNF Receptor SuperfamilyLigands TL1A TNF Receptor Superfamily Ligands CD70 TNF ReceptorSuperfamily Ligands CD30 Ligand TNF Receptor Superfamily Ligands TRAF1TNF Receptor Superfamily Ligands TRAF2 TNF Receptor Superfamily LigandsTRAF3 TNF Receptor Superfamily Ligands TRAIL TNF Receptor SuperfamilyLigands RANK Ligand TNF Receptor Superfamily Ligands APRIL TNF ReceptorSuperfamily Ligands BAFF TNF Receptor Superfamily Ligands B and Tlymphocyte Attenuators TNF Receptor Superfamily Ligands NGF TNF ReceptorSuperfamily Ligands BDNF TNF Receptor Superfamily Ligands Neurotrophin-3TNF Receptor Superfamily Ligands Neurotrophin-4 TNF Receptor SuperfamilyLigands TL6 TNF Receptor Superfamily Ligands Ectodysplasin A2 TNFReceptor Superfamily Ligands Ectodysplasin A1 TNF blockers Remicade(infliximab) TNF blockers Enbrel (etanercept) TNF blockers Humira(adalimumab) TNF blockers Cimzia (certolizumab pegol) TNF blockersSimponi (golimumab) Tumor Necrosis Factor Receptor Family Agonists TollLike Receptors Agonist TIMP-3 Inhibitors BCL-2 Family Inhibitors IAPDisruptors Protease Inhibitors Amino Sugars Chemotherapeutic Cytotoxicchemical Toxins Tyrosine Kinase inhibitors Imatinib Mesylate ProtonsAntivascular Agents Bevacizumab EGFR Inhibitors Erlotinib Anti-InfectiveAgents Antibiotics Anti-Viral Agents Anti-Fungal Agents AminoglycosideStatins Nanoparticles Liposomes Polymers Biopolymers PolysaccharideProteoglycan Glycosaminoglycans Polyethylene glycol Lipids DendrimersFatty Acids Glucocorticoid Corticosteroid Collagenase Inhibitor MatrixMetalloprotease Inhibitors MMP-13 inhibitor Vitamins Vitamin DAntibiotics Antiviral Antifungal Statins Immune Modulators RadioisotopesToxins Enzymes Sensitizing drugs Anti-Angiogenic Agents CisplatinAnti-Angiogenic Agents Anti-Metabolites Anti-Angiogenic Agents MitoticInhibitors Anti-Angiogenic Agents Growth Factor InhibitorsChemotherapeutic Agent Paclitaxel Chemotherapeutic Agent TemozolomideChemotherapeutic Agent Topotecan Chemotherapeutic Agent FluorouracilChemotherapeutic Agent Vincristine Chemotherapeutic Agent VinblastineChemotherapeutic Agent Procarbazine Chemotherapeutic Agent DecarbazineChemotherapeutic Agent Altretamine Chemotherapeutic Agent MethotrexateChemotherapeutic Agent Mercaptopurine Chemotherapeutic Agent ThioguanineChemotherapeutic Agent Fludarabine Phosphate Chemotherapeutic AgentCladribine Chemotherapeutic Agent Pentostatin Chemotherapeutic AgentCytarabine Chemotherapeutic Agent Azacitidine Chemotherapeutic AgentEtoposide Chemotherapeutic Agent Teniposide Chemotherapeutic AgentIrinotecan Chemotherapeutic Agent Docetaxel Chemotherapeutic AgentDoxorubicin Chemotherapeutic Agent Daunorubicin Chemotherapeutic AgentDactinomycin Chemotherapeutic Agent Idarubicin Chemotherapeutic AgentPlicamycin Chemotherapeutic Agent Mitomycin Chemotherapeutic AgentBleomycin Chemotherapeutic Agent Tamoxifen Chemotherapeutic AgentFlutamide Chemotherapeutic Agent Leuprolide Chemotherapeutic AgentGoserelin Chemotherapeutic Agent Aminogluthimide Chemotherapeutic AgentAnastrozole Chemotherapeutic Agent Amsacrine Chemotherapeutic AgentAsparaginase Chemotherapeutic Agent Mitoxantrone Chemotherapeutic AgentMitotane Chemotherapeutic Agent Amifostine Apoptotic Agents Cell Deathor Cell Killing Agents Caspases Apoptosis Activators ApoptosisInhibitors XBP-1 Apoptosis Inhibitors Bcl-2 Apoptosis Inhibitors Bcl-XlApoptosis Inhibitors Bcl-w Nonsteroidal Anti-Inflammatory COX-2Inhibitors Drugs (NSAID) Nonsteroidal Anti-Inflammatory Ketorolac Drugs(NSAID) Nonsteroidal Anti-Inflammatory Indomethacin Drugs (NSAID)Nonsteroidal Anti-Inflammatory Etodolac Drugs (NSAID) NonsteroidalAnti-Inflammatory Tolemetin Drugs (NSAID) Nonsteroidal Anti-InflammatoryNaproxen Drugs (NSAID) Nonsteroidal Anti-Inflammatory Enolic AcidDerivatives Drugs (NSAID) Nonsteroidal Anti-Inflammatory AnthranilicAcid Derivatives Drugs (NSAID) Nonsteroidal Anti-Inflammatory CelecoxibDrugs (NSAID) Nonsteroidal Anti-Inflammatory Sulfonanilides Drugs(NSAID) Nonsteroidal Anti-Inflammatory Salicylates Drugs (NSAID)Nonsteroidal Anti-Inflammatory Aceclofenac Drugs (NSAID) NonsteroidalAnti-Inflammatory Nabumetone Drugs (NSAID) NonsteroidalAnti-Inflammatory Sulindac Drugs (NSAID) Nonsteroidal Anti-InflammatoryDiclofenac Drugs (NSAID) Nonsteroidal Anti-Inflammatory Ibuprofen Drugs(NSAID) Steroids Dexamethasone Steroids Budesonide SteroidsTriamcinolone Steroids Triamcinolone acetonide Steroids CortisoneSteroids Prednisone Steroids Prednisolone Steroids TriamcinoloneHexacetonide Steroids Methylprednisolone Pain Reliever AcetaminophenOpioids Local Anesthetics Anti-Depressants Glutamate ReceptorAntagonists Adenosine Neuropeptides Uricase Elastase

Further examples of active agents include but are not limited to: apeptide, an oligopeptide, a polypeptide, a peptidomimetic, apolynucleotide, a polyribonucleotide, a DNA, a cDNA, a ssDNA, a RNA, adsRNA, a micro RNA, an RNAi, an oligonucleotide, an antibody, a singlechain variable fragment (scFv), an antibody fragment, an aptamer, acytokine, an interferon, a hormone, an enzyme, a growth factor, acheckpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4inhibitor, a CD antigen, aa chemokine, a neurotransmitter, an ionchannel inhibitor, a G-protein coupled receptor inhibitor, a G-proteincoupled receptor activator, a chemical agent, a radiosensitizer, aradioprotectant, a radionuclide, a therapeutic small molecule, asteroid, a corticosteroid, an anti-inflammatory agent, an immunemodulator, a complement fixing peptide or protein, a tumor necrosisfactor inhibitor, a tumor necrosis factor activator, a tumor necrosisfactor receptor family agonist, a tumor necrosis receptor antagonist, atumor necrosis factor (TNF) soluble receptor or antibody, caspaseprotease activator or inhibitor, an NF-κB a RIPK1 and/or RIPK3 inhibitoror activator (e.g., through Toll-like receptors (TLRs) TLR-3 and/orTLR-4, or T-cell receptor (TCR) and the like), a death-receptor ligand(e.g., Fas ligand) activator or inhibitor, TNF receptor family (e.g.,TNFR¹, TNFR², lymphotoxin β receptor/TNFRS3, OX40/TNFRSF4, CD40/TNFRSF5,Fas/TNFRSF6, decoy receptor 3/TNFRSF6B, CD27/TNFRSF7, CD30/TNFRSF8,4-1BB/TNFRSF9, DR⁴ (death receptor 4/TNFRS10A), DR5 (death receptor5/TNFRSF10B), decoy receptor 1/TNFRSF10C, decoy receptor 2/TNFRSF10D,RANK (receptor activator of NF-kappa B/TNFRSF11A), OPG(osteoprotegerin/TNFRSF11B), DR³ (death receptor 3/TNFRSF25), TWEAKreceptor/TNFRSF12A, TAC1/TNFRSF13B, BAFF-R (BAFF receptor/TNFRSF13C),HVEM (herpes virus entry mediator/TNFRSF14), nerve growth factorreceptor/TNFRSF16, BCMA (B cell maturation antigen/TNFRSF17), GITR(glucocorticoid-induced TNF receptor/TNFRSF18), TAJ (toxicity and JNKinducer/TNFRSF19), RELT/TNFRSF19L, DR⁶ (death receptor 6/TNFRSF21),TNFRSF22, TNFRSF23, ectodysplasin A² isoform receptor/TNFRS27,ectodysplasin 1, and anhidrotic receptor, a TNF receptor superfamilyligand including—TNF alpha, lymphotoxin-α, tumor necrosis factormembrane form, tumor necrosis factor shed form, LIGHT, lymphotoxin β₂α₁heterotrimer, OX-40 ligand, compound 1 [PMID: 24930776], CD40 ligand,Fas ligand, TL1A, CD70, CD30 ligand, TRAF1, TRAF2, TRAF3, TRAIL, RANKligand, APRIL, BAFF, B and T lymphocyte attenuator, NGF, BDNF,neurotrophin-3, neurotrophin-4, TL6, ectodysplasin A², ectodysplasinA1—a TIMP-3 inhibitor, a BCL-2 family inhibitor, navitoclax (Aging Cell.15(3): 428-435. (2016)) an IAP disruptor, a protease inhibitor, an aminosugar, a chemotherapeutic (whether acting through an apoptotic ornon-apoptotic pathway) (Ricci et al. Oncologist 11(4):342-57 (2006)), acytotoxic chemical, a toxin, a tyrosine kinase inhibitor (e.g., imatinibmesylate), protons, bevacuzimab (antivascular agent), erlotinib (EGFRinhibitor), an anti-infective agent, an antibiotic, an anti-viral agent,an anti-fungal agent, an aminoglycoside, a nonsteroidalanti-inflammatory drug (NSAID), a statin, a nanoparticle, a liposome, apolymer, a biopolymer, a polysaccharide, a proteoglycan, aglycosaminoglycan, polyethylene glycol, a lipid, a dendrimer, a fattyacid, or an Fc domain or an Fc region, or an active fragment or amodification thereof. Any combination of the above active agents can beco-delivered with peptides or peptide conjugates of this disclosure.Additionally, in some embodiments, other co-therapies such as protontherapy or ablative radiotherapy can be administered to a subject inneed thereof along with peptides or peptide conjugates of thisdisclosure. In some embodiments, the peptide is covalently ornon-covalently linked to an active agent, e.g., directly or via alinker. TNF blockers suppress the immune system by blocking the activityof TNF, a substance in the body that can cause inflammation and lead toimmune-system diseases, such as Crohn's disease, ulcerative colitis,rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis andplaque psoriasis. The drugs in this class include Remicade (infliximab),Enbrel (etanercept), Humira (adalimumab), Cimzia (certolizumab pegol)and Simponi (golimumab). The peptide disclosed herein can be used tohome, distribute to, target, directed to, is retained by, accumulate in,migrate to, and/or bind to cartilage, and thus also be used forlocalizing the attached or fused active agent. Furthermore,cystine-dense chlorotoxin peptide can be internalized in cells(Wiranowska, M. , Cancer Cell Int. , 11: 27 (2011)). Therefore, cellularinternalization, subcellular localization, and intracellular traffickingafter internalization of the peptide itself, or an active agent peptideconjugate or fusion peptide can be important factors in the efficacy ofan active agent conjugate or fusion. (Ducry, L., Antibody DrugConjugates (2013); and Singh, S. K., Pharm Res., 32(11): 3541-3571(2015)). Exemplary linkers suitable for use with the embodiments hereinare discussed in further detail below.

The peptides or peptide-active agent fusions of the present disclosurecan also be conjugated to other moieties that can serve other roles,such as providing an affinity handle (e.g., biotin) for retrieval of thepeptides from tissues or fluids. For example, peptides or peptide-activeagent fusions of the present disclosure can also be conjugated tobiotin. In addition to extension of half-life, biotin could also act asan affinity handle for retrieval of peptides or peptide-active agentfusions from tissues or other locations. In some embodiments,fluorescent biotin conjugates that can act both as a detectable labeland an affinity handle can be used. Non limiting examples ofcommercially available fluorescent biotin conjugates include Atto425-Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto740-Biotin, fluorescein biotin, biotin-4-fluorescein,biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, Alexafluor 488 biocytin, Alexa flour 546, Alexa Fluor 549, lucifer yellowcadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin,biotin-rhodamine and tetramethylrhodamine biocytin. In some otherexamples, the conjugates could include chemiluminescent compounds,colloidal metals, luminescent compounds, enzymes, radioisotopes, andparamagnetic labels. In some embodiments, the peptide-active agentfusions described herein can be attached to another molecule. Forexample, the peptide sequence also can be attached to another activeagent (e.g., small molecule, peptide, polypeptide, polynucleotide,antibody, aptamer, cytokine, growth factor, neurotransmitter, an activefragment or modification of any of the preceding, fluorophore,radioisotope, radionuclide chelator, acyl adduct, chemical linker, orsugar, etc.). In some embodiments, the peptide can be fused with, orcovalently or non-covalently linked to an active agent.

Additionally, more than one peptide sequence can be present on or fusedwith a particular peptide. A peptide can be incorporated into abiomolecule by various techniques, for example by a chemicaltransformation, such as the formation of a covalent bond, such as anamide bond, or by solid phase or solution phase peptide synthesis, or bypreparing a nucleic acid sequence encoding the biomolecule, wherein thenucleic acid sequence includes a subsequence that encodes the peptide.The subsequence can be in addition to the sequence that encodes thebiomolecule, or can substitute for a subsequence of the sequence thatencodes the biomolecule.

Detectable Agent Conjugates

Described herein are agents that can be conjugated to the peptides ofthe present invention for use in detection and tracing either cartilagedisorders or kidney disorders, or both. As described herein, it isunderstood that certain active agents are described in a non-limitingexemplary manner for use in diagnostics, aiding surgery and treatment,prognosis and tracking of progress or remission of cartilage and/orkidney disorders, diseases or injury. One or more of such detectableagents can be conjugated to a peptide of the present invention alone orin combination with one or more active agents described herein. Moreoversome detectable agents (e.g., radionuclides, radioisotopes,radiosensitizers and photosensitizers amongst others) may also exerttherapeutic activity as well. A peptide can be conjugated to an agentused in imaging, research, therapeutics, theranostics, pharmaceuticals,chemotherapy, chelation therapy, targeted drug delivery, andradiotherapy. The agent can be a detectable agent. In some embodiments,a peptide of the present invention is conjugated to detectable agents,such as a metal, a radioisotope, a dye, fluorophore, or another suitablematerial that can be used in imaging. Non-limiting examples ofradioisotopes include alpha emitters, beta emitters, positron emitters,and gamma emitters. In some embodiments, the metal or radioisotope isselected from the group consisting of actinium, americium, bismuth,cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium,manganese, palladium, polonium, radium, ruthenium, samarium, strontium,technetium, thallium, and yttrium. In some embodiments, the metal isactinium, bismuth, lead, radium, strontium, samarium, or yttrium. Insome embodiments, the radioisotope is actinium-225 or lead-212. In someembodiments, the fluorophore is a fluorescent agent emittingelectromagnetic radiation at a wavelength between 650 nm and 4000 nm,such emissions being used to detect such agent. In some embodiments thefluorophore is a fluorescent agent is selected from the group consistingof non-limiting examples of fluorescent dyes that could be used as aconjugating molecule (or as applied to each class of molecules) in thepresent disclosure include DyLight-680, DyLight-750, VivoTag-750,DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICGclass of dyes). In some embodiments, near infrared dyes include cyaninedyes. Additional non-limiting examples of fluorescent dyes for use as aconjugating molecule in the present disclosure include acradine orangeor yellow, Alexa Fluors and any derivative thereof, 7-actinomycin D,8-anilinonaphthalene-1-sulfonic acid, ATTO dye and any derivativethereof, auramine-rhodamine stain and any derivative thereof,bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene,5,12-bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein,carbodyfluorescein and any derivative thereof,1-chloro-9,10-bis(phenylethynyl)anthracene and any derivative thereof,DAPI, DiOC6, DyLight Fluors and any derivative thereof, epicocconone,ethidium bromide, FlAsH-EDT₂, Fluo dye and any derivative thereof,FluoProbe and any derivative thereof, Fluorescein and any derivativethereof, Fura and any derivative thereof, GelGreen and any derivativethereof, GelRed and any derivative thereof, fluorescent proteins and anyderivative thereof, m isoform proteins and any derivative thereof suchas for example mCherry, hetamethine dye and any derivative thereof,hoeschst stain, iminocoumarin, indian yellow, indo-1 and any derivativethereof, laurdan, lucifer yellow and any derivative thereof, luciferinand any derivative thereof, luciferase and any derivative thereof,mercocyanine and any derivative thereof, nile dyes and any derivativethereof, perylene, phloxine, phyco dye and any derivative thereof,propium iodide, pyranine, rhodamine and any derivative thereof,ribogreen, RoGFP, rubrene, stilbene and any derivative thereof,sulforhodamine and any derivative thereof, SYBR and any derivativethereof, synapto-pHluorin, tetraphenyl butadiene, tetrasodium tris,Texas Red, Titan Yellow, TSQ, umbelliferone, violanthrone, yellowfluorescent protein and YOYO-1. Other Suitable fluorescent dyes include,but are not limited to, fluorescein and fluorescein dyes (e.g.,fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 6-carboxyfluorescein or FAM,etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes,phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g.,carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.),Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes(e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXAFLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXAFLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXAFLUOR 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R⁶G, BODIPY TMR,BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, etc.), IRDyes(e.g., IRD40, IRD 700, IRD 800, etc.), indocyanine green dyes and thelike. For each of the above listed fluorescent dyes various activatedforms can be used for conjugation. Additional suitable detectable agentsare described in PCT/US14/56177. Non-limiting examples of radioisotopesinclude alpha emitters, beta emitters, positron emitters, and gammaemitters. In some embodiments, the metal or radioisotope is selectedfrom the group consisting of actinium, americium, bismuth, cadmium,cesium, cobalt, europium, gadolinium, iridium, lead, lutetium,manganese, palladium, polonium, radium, ruthenium, samarium, strontium,technetium, thallium, and yttrium. In some embodiments, the metal isactinium, bismuth, lead, radium, strontium, samarium, or yttrium. Insome embodiments, the radioisotope is actinium-225 or lead-212.

Other embodiments of the present disclosure provide peptides conjugatedto a radiosensitizer or photosensitizer. Examples of radiosensitizersinclude but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel,carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole,misonidazole, tirapazamine, and nucleic acid base derivatives (e.g.,halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine).Examples of photosensitizers include but are not limited to: fluorescentmolecules or beads that generate heat when illuminated, porphyrins andporphyrin derivatives (e.g., chlorins, bacteriochlorins,isobacteriochlorins, phthalocyanines, and naphthalocyanines),metalloporphyrins, metallophthalocyanines, angelicins,chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins and relatedcompounds such as alloxazine and riboflavin, fullerenes, pheophorbides,pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins,sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums,methylene blue derivatives, naphthalimides, nile blue derivatives,quinones, perylenequinones (e.g., hypericins, hypocrellins, andcercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes,verdins, xanthene dyes (e.g., eosins, erythrosins, rose bengals),dimeric and oligomeric forms of porphyrins, and prodrugs such as5-aminolevulinic acid. Advantageously, this approach allows for highlyspecific targeting of diseased cells (e.g., cancer cells) using both atherapeutic agent (e.g., drug) and electromagnetic energy (e.g.,radiation or light) concurrently. In some embodiments, the peptide iscovalently or non-covalently linked to the agent, e.g., directly or viaa linker. Exemplary linkers suitable for use with the embodiments hereinare discussed in further detail below.

Linkers

Peptides according to the present disclosure that home, target, migrateto, are retained by, accumulate in, and/or bind to, or are directed tothe cartilage can be attached to another moiety (e.g., an active agent),such as a small molecule, a second peptide, a protein, an antibody, anantibody fragment, an aptamer, polypeptide, polynucleotide, afluorophore, a radioisotope, a radionuclide chelator, a polymer, abiopolymer, a fatty acid, an acyl adduct, a chemical linker, or sugar orother active agent described herein through a linker, or directly in theabsence of a linker.

A peptide can be directly attached to another molecule by a covalentattachment. For example, the peptide is attached to a terminus of theamino acid sequence of a larger polypeptide or peptide molecule, or isattached to a side chain, such as the side chain of a lysine, serine,threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acidresidue, or glutamic acid residue. The attachment can be via an amidebond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogenbond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, acarbon-carbon single double or triple bond, a disulfide bond, or athioether bond. In some embodiments, similar regions of the disclosedpeptide(s) itself (such as a terminus of the amino acid sequence, anamino acid side chain, such as the side chain of a lysine, serine,threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acidresidue, or glutamic acid residue, via an amide bond, an ester bond, anether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, amacrocycle, an oxime bond, a hydrazone bond, a carbon-carbon singledouble or triple bond, a disulfide bond, or a thioether bond, or linkeras described herein) can be used to link other molecules.

Attachment via a linker can involve incorporation of a linker moietybetween the other molecule and the peptide. The peptide and the othermolecule can both be covalently attached to the linker. The linker canbe cleavable, labile, non-cleavable, stable self-immolating,hydrophilic, or hydrophobic. As used herein, the term “non-cleavable”(such as used in association with an amide, cyclic, or carbamate linkeror as otherwise as described herein) is often used by a skilled artisanto distinguish a relatively stable structure from one that is morelabile or “cleavable” (e.g., as used in association with cleavablelinkers that may be dissociated or cleaved structurally by enzymes,proteases, self-immolation, pH, reduction, hydrolysis, certainphysiologic conditions, or as otherwise described herein). It isunderstood that “non-cleavable” linkers offer stability against cleavageor other dissociation as compared to “cleavable” linkers, and the termis not intended to be considered an absolute non-cleavable ornon-dissociative structure under any conditions. Consequently, as usedherein, a “non-cleavable” linker is also referred to as a “stable”linker. The linker can have at least two functional groups with onebonded to the peptide, the other bonded to the other molecule, and alinking portion between the two functional groups.

Non-limiting examples of the functional groups for attachment caninclude functional groups capable of forming an amide bond, an esterbond, an ether bond, a carbonate bond, a carbamate bond, or a thioetherbond. Non-limiting examples of functional groups capable of forming suchbonds can include amino groups; carboxyl groups; hydroxyl groups;aldehyde groups; azide groups; alkyne and alkene groups; ketones;hydrazides; acid halides such as acid fluorides, chlorides, bromides,and iodides; acid anhydrides, including symmetrical, mixed, and cyclicanhydrides; carbonates; carbonyl functionalities bonded to leavinggroups such as cyano, succinimidyl, and N-hydroxysuccinimidyl; hydroxylgroups; sulfhydryl groups; and molecules possessing, for example, alkyl,alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides,mesylates, tosylates, triflates, epoxides, phosphate esters, sulfateesters, and besylates.

Non-limiting examples of the linking portion can include alkylene,alkenylene, alkynylene, polyether, such as polyethylene glycol (PEG),hydroxy carboxylic acids, polyester, polyamide, polyamino acids,polypeptides, cleavable peptides, valine-citrulline,aminobenzylcarbamates, D-amino acids, and polyamine, any of which beingunsubstituted or substituted with any number of substituents, such ashalogens, hydroxyl groups, sulfhydryl groups, amino groups, nitrogroups, nitroso groups, cyano groups, azido groups, sulfoxide groups,sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehydegroups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups,halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups,aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups,heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups,amide groups, urethane groups, epoxides, and ester groups.

A peptide and drug conjugated via a linker is described with the formulaPeptide-A-B-C-Drug, wherein the linker is A-B-C. A can be a stable amidelink, is an amine on the peptide and the linker and can be achieved viaa tetrafluorophenyl (TFP) ester or an NHS ester. B can be (—CH₂—)_(x)—or a short PEG (—CH₂CH₂O—)_(x) (x is 1-10), and C can be the ester bondto the hydroxyl or carboxylic acid on the drug. In some embodiments, Ccan refer to the “cleavable” or “stable” part of the linker. In otherembodiments, A can also be the “cleavable” part. In some embodiments, Acan be amide, carbamate, thioether via maleimide or bromoacetamide,triazole, oxime, or oxacarboline. The cleaved active agent or drug canretain the chemical structure of the active agent before cleavage, orcan be modified as a result of cleavage. Moreover, depending on thedesired therapeutic properties of the peptide-drug conjugate, suchactive agent can be active while linked to the peptide, remain activeafter cleavage or become inactivated, be inactive while linked to thepeptide, or it can be activated upon cleavage.

In some embodiments, peptide conjugates have stable linkers. A peptideof the disclosure can be expressed recombinantly or chemicallysynthesized. The peptide can be conjugated to a detectable agent or anactive agent via a stable linker, such as an amide linkage or acarbamate linkage. The peptide can be conjugated to a detectable agentor an active agent via a stable linker, such as an amide bond usingstandard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) ordicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride orphosphorous chloride-based bioconjugation chemistries. A stable linkermay or may not be cleaved in buffer over extended periods of time (e.g.,hours, days, or weeks). A stable linker may or may not be cleaved inbody fluids such as plasma or synovial fluid over extended periods oftime (e.g., hours, days, or weeks). A stable linker, may or may not becleaved after exposure to enzymes, reactive oxygen species, otherchemicals or enzymes that can be present in cells (e.g., macrophages),cellular compartments (e.g., endosomes and lysosomes), inflamed areas ofthe body (e.g., inflamed joints), tissues or body compartments. A stablelinker may be cleaved by unknown mechanisms. A stable linker may or maynot be cleaved in vivo but remains an active agent after peptideconjugation.

A peptide and drug conjugated via a linker can be described with theformula Peptide-A-B-C-Drug, wherein the linker is A-B-C. A can be astable amide link such as that formed by reacting an amine on thepeptide with a linker containing a tetrafluorophenyl (TFP) ester or anNHS ester. A can also be a stable carbamate linker such as that formedby reacting an amine on the peptide with an imidazole carbamate activeintermediate formed by reaction of CDI with a hydroxyl on the linker. Acan also be a stable secondary amine linkage such as that formed byreductive alkylation of the amine on the peptide with an aldehyde orketone group on the linker. A can also be a stable thioether linkerformed using a maleimide or bromoacetamide in the linker with a thiol inthe peptide, a triazole linker, a stable oxime linker, or a oxacarbolinelinker. B can be (—CH2—)_(x)— or a short PEG (—CH₂CH₂O—)_(x) (x is 0-20)or other spacers or no spacer. C can be an amide bond formed with anamine or a carboxylic acid on the drug, a thioether formed between amaleimide on the linker and a sulihydroyl on the drug, a secondary ortertiary amine, a carbamate, or other stable bonds. Any linker chemistrydescribed in “Current ADC Linker Chemistry,” Jain et al. , Pharm Res,2015 DOI 10. 1007/s11095-015-1657-7 can be used.

The resulting peptide conjugates can be administered to a human oranimal subcutaneously, intravenously, orally, or injected directly intoa joint to treat disease. The peptide is not specifically cleaved fromthe detectable agent or active agent via a targeted mechanism. Thepeptide can be degraded by mechanisms such as catabolism, releasing adrug that is modified or not modified form its native form(Antibody-Drug Conjugates: Design, Formulation, and PhysicochemicalStability, Singh, Luisi, and Pak. Pharm Res (2015) 32:3541-3571). Thepeptide drug conjugate exerts its pharmacological activity while stillintact, or while partially or fully degraded, metabolized, orcatabolized.

In some embodiments, peptide conjugates can have cleavable linkers. Insome embodiments, a peptide and drug can be conjugated via a linker andcan be described with the formula Peptide-A-B-C-Drug, wherein the linkeris A-B-C. In some embodiments, A can be a stable amide link such as thatformed by reacting an amine on the peptide with a linker containing atetrafluorophenyl (TFP) ester or an NHS ester. In certain embodiments, Acan also be a stable carbamate linker that is formed by an aminereaction on the peptide with an imidazole carbamate active intermediateformed by reaction of CDI with a hydroxyl on the linker. In otherembodiments, A can also be a stable secondary amine linkage such as thatformed by reductive alkylation of the amine on the peptide with analdehyde or ketone group on the linker. In some embodiments, A can alsobe a stable thioether linker formed using a maleimide or bromoacetamidein the linker with a thiol in the peptide, a triazole linker, a stableoxime linker, or an oxacarboline linker. B can be (—CH2—)_(x)— or ashort PEG (—CH₂CH₂O—)_(x) (x is 0-20) or other spacers or no spacer. Ccan be an ester bond to the hydroxyl or carboxylic acid on the drug, ora carbonate, hydrazone, or acylhydrazone, designed for hydrolyticcleavage. The hydrolytic rate of cleavage can be varied by varying thelocal environment around the bond, including carbon length (—CH2—)_(x),steric hindrance (including adjacent side groups such as methyl, ethyl,cyclic), hydrophilicity or hydrophobicity. In some embodiments, peptideconjugates can have a linear or cyclic ester linkage, which can includeor do not include side chains such as methyl or ethyl groups. A linearester linkage can be more susceptible to cleavage (such as byhydrolysis, an enzyme such as esterase, or other chemical reaction) thana cyclic ester due to steric hindrance or hydrophobicity/hydrophilicityeffects. Likewise, side chains such as methyl or ethyl groups on thelinear ester linkage can optionally make the linkage less susceptible tocleavage than without the side chains. In some embodiments, hydrolysisrate can be affected by local pH, such as lower pH in certaincompartments of the body or of the cell such as endosomes and lysosomesor diseased tissues. In some embodiments, C can also be a pH sensitivegroup such as a hydrazone or oxime linkage. In other embodiments, C canbe a disulfide bond designed to be released by reduction, such as byglutathione. In other embodiments, (or A-B-C) can be a peptidic linkagedesign for cleavabe by enzymes. Optionally, a self-immolating group suchas pABC can be included to cause release of a free unmodified drug uponcleavage (Antibody-Drug Conjugates: Design, Formulation, andPhysicochemical Stability, Singh, Luisi, and Pak. Pharm Res (2015)32:3541-3571). The linker can be cleaved by enzymes such as esterases,matrix metalloproteinases, cathepsins such as cathepsin B,glucuronidases, a protease, or thrombin. Alternatively, the bonddesigned for cleavage can be at A, rather than C, and C can be a stablebond or a cleavable bond. An alternative design can be to have stablelinkers (such as amide or carbamate) at A and C and have a cleavablelinker in B, such as a disulfide bond. The rate of reduction can bemodulated by local effects such as steric hindrance from methyl or ethylgroups or modulating hydrophobicity/hydrophilicity. In some embodiments,peptide conjugates can have an ester carbonyl linkage, a longhydrocarbon linker, or carbamate linker, each of which can includehydrophilic groups, such as alcohols, acids, or ethers, or include ahydrocarbon side chain or other moiety that tunes the rate of cleavage.For example, the rate of hydrolysis can be faster with hydrophilicgroups, such as alcohols, acids, or ethers, near an ester carbonyl. Inanother example, hydrophobic groups present as side chains or as alonger hydrocarbon linker can slow the cleavage rate of the ester.Likewise, cleavage of a carbamate group can also be tuned by hindrance,hydrophobicity, and the like. In another example, using a less labilelinking group, such as a carbamate rather than an ester, can slow thecleavage rate of the linker.

Non-limiting examples of linkers include:

wherein each n is independently 0 to about 1,000; 1 to about 1,000; 0 toabout 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 toabout 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1to about 10; 0 to about 5; or 1 to about 5. In some embodiments, each nis independently 0, about 1, about 2, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, about 19, about 20,about 21, about 22, about 23, about 24, about 25, about 26, about 27,about 28, about 29, about 30, about 31, about 32, about 33, about 34,about 35, about 36, about 37, about 38, about 39, about 40, about 41,about 42, about 43, about 44, about 45, about 46, about 47, about 48,about 49, or about 50. In some embodiments, m is 1 to about 1,000; 1 toabout 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 toabout 20; 1 to about 15; 1 to about 10; or 1 to about 5. In someembodiments, m is 0, about 1, about 2, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, about 19, about 20,about 21, about 22, about 23, about 24, about 25, about 26, about 27,about 28, about 29, about 30, about 31, about 32, about 33, about 34,about 35, about 36, about 37, about 38, about 39, about 40, about 41,about 42, about 43, about 44, about 45, about 46, about 47, about 48,about 49, or about 50.

In some cases a linker can be a succinic linker, and a drug can beattached to a peptide via an ester bond or an amide bond with twomethylene carbons in between. In other cases, a linker can be any linkerwith both a hydroxyl group and a carboxylic acid, such as hydroxyhexanoic acid or lactic acid.

The linker can be a cleavable or a stable linker. The use of a cleavablelinker permits release of the conjugated moiety (e.g., a therapeuticagent) from the peptide, e.g., after targeting to the cartilage. In somecases the linker is enzyme cleavable, e.g., a valine-citrulline linker.In some embodiments, the linker contains a self-immolating portion. Inother embodiments, the linker includes one or more cleavage sites for aspecific protease, such as a cleavage site for matrix metalloproteases(MMPs), thrombin, or cathepsin. Alternatively or in combination, thelinker is cleavable by other mechanisms, such as via pH, reduction, orhydrolysis. A hydrolytically labile linker, (amongst other cleavablelinkers described herein) can be advantageous in terms of releasingactive agents from the peptide. For example, an active agent in aconjugate form with the peptide may not be active, but upon release fromthe conjugate after targeting to the cartilage, the active agent isactive.

The rate of hydrolysis of the linker can be tuned. For example, the rateof hydrolysis of linkers with unhindered esters is faster compared tothe hydrolysis of linkers with bulky groups next an ester carbonyl. Abulky group can be a methyl group, an ethyl group, a phenyl group, aring, or an isopropyl group, or any group that provides steric bulk. Insome cases, the steric bulk can be provided by the drug itself, such asby ketorolac when conjugated via its carboxylic acid. The rate ofhydrolysis of the linker can be tuned according to the residency time ofthe conjugate in the cartilage. For example, when a peptide is clearedfrom the cartilage relatively quickly, the linker can be tuned torapidly hydrolyze. In contrast, for example, when a peptide has a longerresidence time in the cartilage, a slower hydrolysis rate can allow forextended delivery of an active agent. This can be important when thepeptide is used to deliver a drug to the cartilage. “Programmedhydrolysis in designing paclitaxel prodrug for nanocarrier assembly” SciRep 2015, 5, 12023 Fu et al. , provides an example of modifiedhydrolysis rates.

Peptide Stability

A peptide of the present disclosure can be stable in various biologicalconditions, as well as during manufacturing, handling, storage, andother conditions in either a liquid or a dried state. Additionally, apeptide of the present disclosure can be resistant to enzymatic cleavageneeded for peptide processing by the immune system. For example, anypeptide of SEQ ID NO: 485—SEQ ID NO: 1048 can exhibit resistance toreducing agents, proteases, oxidative conditions, or acidic conditions.

In some cases, biologic molecules (such as peptides and proteins) canprovide therapeutic functions, but such therapeutic functions aredecreased or impeded by instability caused by the in vivo environment.(Moroz et al., Adv Drug Deliv Rev 101:108-21 (2016), Mitragotri et al.,Nat Rev Drug Discov 13(9):655-72 (2014), Bruno et al., Ther Deliv(11):1443-67 (2013), Sinha et al., Crit Rev Ther Drug Carrier Syst.24(1):63-92 (2007), Hamman et al., BioDrugs 19(3):165-77 (2005)).Peptide degradation can be a result of a number of processes involvinghydrolytic pathways, peptide oxidation such as oxidation of methionine(Met) residues, deamidation of asparagine (Asn) and glutamine (Gln)residues, and isomerization and hydrolysis of an adjacent asparagine(Asp) residue. (Manning et al., Pharmaceutical Research, Vol. 27 No. 4(2010)). The amino acid immediately following the Asn or Gln residue canalso affect the rate of deamidation, whereas: Asn-Gly, Asn-Ser, Asn-His,and Gln-Gly can be more likely to undergo deamidation. Additionally, thepeptide bond adjacent to amino acids such as Asp can undergo hydrolysiswith amino acid pairings such as Asp-Gly, Asp-Ser, Asp-Tyr, and Asp-Pro,which can be more likely to undergo hydrolysis. Oxidation of amino acidresidues such as Met can form a sulfoxide species. The specificdegradation reactions rates can vary for any given peptide or proteinsequence.

Furthermore, the microenvironment within the molecular structure of thepeptide, solvent accessibility, and conformational stability of eachresidue can impact the likelihood of peptide degradation. Therefore, bymodifying a peptide sequence to reduce occurrence of such degradationevents, a the modified peptide or peptide-conjugate can have increasedbeneficial properties over unmodified peptides or peptide-drugconjugates, such as improved therapeutic efficacy, an increased safetyprofile, and can be less expensive to manufacture and develop. Keyformulaic considerations that can prevent peptide decay can include theuse of excipients, formulation at a desired pH, and storage underspecific conditions (e.g., temperature, oxygen, light exposure, solid orliquid state, and container excipient materials). To circumventdegradation, peptide residues can be substituted with amino acids thatincrease stability, which can result in more efficacious and durabletherapeutic peptides.

With respect to in vivo stability, the GI tract can contain a region oflow pH (e.g., pH ˜1), a reducing environment, or a protease-richenvironment that can degrade peptides and proteins. Proteolytic activityin other areas of the body, such as the mouth, eye, lung, intranasalcavity, joint, skin, vaginal tract, mucous membranes, and serum, canalso be an obstacle to the delivery of functionally active peptides andpolypeptides. Additionally, the half-life of peptides in serum can bevery short, in part due to proteases, such that the peptide can bedegraded too quickly to have a lasting therapeutic effect whenadministering a therapeutic and safe dosing regimen. Likewise,proteolytic activity in cellular compartments, such as lysosomes, andreduction activity in lysosomes and the cytosol can degrade peptides andproteins such that they may be unable to provide a therapeutic functionon intracellular targets. Therefore, peptides that are resistant toreducing agents, proteases, and low pH may be able to provide enhancedtherapeutic effects or enhance the therapeutic efficacy of co-formulatedor conjugated active agents in vivo.

Additionally, oral delivery of drugs can be desirable in order to targetcertain areas of the body (e.g., disease in the GI tract such as coloncancer, irritable bowel disorder, infections, metabolic disorders, andconstipation) despite the obstacles to the delivery of functionallyactive peptides and polypeptides presented by this method ofadministration. For example, oral delivery of drugs can increasecompliance by providing a dosage form that is more convenient forpatients to take as compared to parenteral delivery. Oral delivery canbe useful in treatment regimens that have a large therapeutic window.Therefore, peptides that are resistant to reducing agents, proteases,and low pH can allow for oral delivery of peptides without nullifyingtheir therapeutic function.

Peptide Resistance to Reducing Agents. Peptides of this disclosure cancontain one or more cysteines, which can participate in disulfidebridges that can be integral to preserving the folded state of thepeptide. Exposure of peptides to biological environments with reducingagents can result in unfolding of the peptide and loss of functionalityand bioactivity. For example, glutathione (GSH) is a reducing agent thatcan be present in many areas of the body and in cells, and can reducedisulfide bonds. As another example, a peptide can become reduced uponcellular internalization during trafficking of a peptide across thegastrointestinal epithelium after oral administration a peptide canbecome reduced upon exposure to various parts of the GI tract. The GItract can be a reducing environment, which can inhibit the ability oftherapeutic molecules with disulfide bonds to have optimal therapeuticefficacy, due to reduction of the disulfide bonds. A peptide can also bereduced upon entry into a cell, such as after internalization byendosomes or lysosomes or into the cytosol, or other cellularcompartments. Reduction of the disulfide bonds and unfolding of thepeptide can lead to loss of functionality or affect key pharmacokineticparameters such as bioavailability, peak plasma concentration,bioactivity, and half-life. Reduction of the disulfide bonds can alsolead to increased susceptibility of the peptide to subsequentdegradation by proteases, resulting in rapid loss of intact peptideafter administration. In some embodiments, a peptide that is resistantto reduction can remain intact and can impart a functional activity fora longer period of time in various compartments of the body and incells, as compared to a peptide that is more readily reduced.

In certain embodiments, the peptides of this disclosure can be analyzedfor the characteristic of resistance to reducing agents to identifystable peptides. In some embodiments, the peptides of this disclosurecan remain intact after being exposed to different molarities ofreducing agents such as 0.00001M-0.0001M, 0.0001M-0.001M, 0.001M-0.01M,0.01 M-0.05 M, 0.05 M-0.1 M, for greater 15 minutes or more. In someembodiments, the reducing agent used to determine peptide stability canbe dithiothreitol (DTT), Tris (2-carboxyethyl) phosphine HCl (TCEP),2-Mercaptoethanol, (reduced) glutathione (GSH), or any combinationthereof In some embodiments, at least 5%-10%, at least 10%-20%, at least20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of thepeptide remains intact after exposure to a reducing agent.

Peptide Resistance to Proteases. The stability of peptides of thisdisclosure can be determined by resistance to degradation by proteases.Proteases, also referred to as peptidases or proteinases, can be enzymesthat can degrade peptides and proteins by breaking bonds betweenadjacent amino acids. Families of proteases with specificity fortargeting specific amino acids can include serine proteases, cysteineproteases, threonine proteases, aspartic proteases, glutamic proteases,esterases, serum proteases, and asparagine proteases. Additionally,metalloproteases, matrix metalloproteases, elastase, carboxypeptidases,Cytochrome P450 enzymes, and cathepsins can also digest peptides andproteins. Proteases can be present at high concentration in blood, inmucous membranes, lungs, skin, the GI tract, the mouth, nose, eye, andin compartments of the cell. Misregulation of proteases can also bepresent in various diseases such as rheumatoid arthritis and otherimmune disorders. Degradation by proteases can reduce bioavailability,biodistribution, half-life, and bioactivity of therapeutic moleculessuch that they are unable to perform their therapeutic function. In someembodiments, peptides that are resistant to proteases can better providetherapeutic activity at reasonably tolerated concentrations in vivo.

In some embodiments, peptides of this disclosure can resist degradationby any class of protease. In certain embodiments, peptides of thisdisclosure resist degradation by pepsin (which can be found in thestomach), trypsin (which can be found in the duodenum), serum proteases,or any combination thereof. In certain embodiments, peptides of thisdisclosure can resist degradation by lung proteases (e.g., serine,cysteinyl, and aspartyl proteases, metalloproteases, neutrophilelastase, alpha-1 antitrypsin, secretory leucoprotease inhibitor,elafin), or any combination thereof. In some embodiments, the proteasesused to determine peptide stability can be pepsin, trypsin,chymotrypsin, or any combination thereof. In some embodiments, at least5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least80%-90%, or at least 90%-100% of the peptide remains intact afterexposure to a protease. Peptides of SEQ ID NO: 683, SEQ ID NO: 511, andSEQ ID NO: 592 can have particular structural qualities, which make themmore resistant to protease degradation. For example, peptide of SEQ IDNO: 511 and SEQ ID NO: 593 exhibit the “hitchin” topology as describedpreviously, which can be associated with resistance to protease andchemical degradation.

Peptide Stability in Acidic Conditions. Peptides of this disclosure canbe administered in biological environments that are acidic. For example,after oral administration, peptides can experience acidic environmentalconditions in the gastric fluids of the stomach and gastrointestinal(GI) tract. The pH of the stomach can range from ˜1-4 and the pH of theGI tract ranges from acidic to normal physiological pH descending fromthe upper GI tract to the colon. In addition, the vagina, lateendosomes, and lysosomes can also have acidic pH values, such as lessthan pH 7. These acidic conditions can lead to denaturation of peptidesand proteins into unfolded states. Unfolding of peptides and proteinscan lead to increased susceptibility to subsequent digestion by otherenzymes as well as loss of biological activity of the peptide.

In certain embodiments, the peptides of this disclosure can resistdenaturation and degradation in acidic conditions and in buffers, whichsimulate acidic conditions. In certain embodiments, peptides of thisdisclosure can resist denaturation or degradation in buffer with a pHless than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pHless than 5, a pH less than 6, a pH less than 7, or a pH less than 8. Insome embodiments, peptides of this disclosure remain intact at a pH of1-3. In certain embodiments, at least 5%-10%, at least 10%-20%, at least20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of thepeptide remains intact after exposure to a buffer with a pH less than 1,a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5,a pH less than 6, a pH less than 7, or a pH less than 8. In otherembodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, atleast 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, atleast 70%-80%, at least 80%-90%, or at least 90%-100% of the peptideremains intact after exposure to a buffer with a pH of 1-3. In otherembodiments, the peptides of this disclosure can be resistant todenaturation or degradation in simulated gastric fluid (pH 1-2). In someembodiments, at least 5-10%, at least 10%-20%, at least 20%-30%, atleast 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, atleast 70%-80%, at least 80%-90%, or at least 90-100% of the peptideremains intact after exposure to simulated gastric fluid. In someembodiments, low pH solutions such as simulated gastric fluid or citratebuffers can be used to determine peptide stability.

Peptide Stability at High Temperatures. Peptides of this disclosure canbe administered in biological environments with high temperatures. Forexample, after oral administration, peptides can experience hightemperatures in the body. Body temperature can range from 36° C. to 40°C. High temperatures can lead to denaturation of peptides and proteinsinto unfolded states. Unfolding of peptides and proteins can lead toincreased susceptibility to subsequent digestion by other enzymes aswell as loss of biological activity of the peptide. In some embodiments,a peptide of this disclosure can remain intact at temperatures from 25°C. to 100° C. High temperatures can lead to faster degradation ofpeptides. Stability at a higher temperature can allow for storage of thepeptide in tropical environments or areas where access to refrigerationis limited. In certain embodiments, 5%-100% of the peptide can remainintact after exposure to 25° C. for 6 months to 5 years. 5%-100% of apeptide can remain intact after exposure to 70° C. for 15 minutes to 1hour. 5%-100% of a peptide can remain intact after exposure to 100° C.for 15 minutes to 1 hour. In other embodiments, at least 5%-10%, atleast 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, atleast 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, orat least 90%-100% of the peptide remains intact after exposure to 25° C.for 6 months to 5 years. In other embodiments, at least 5%-10%, at least10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or atleast 90%-100% of the peptide remains intact after exposure to 70° C.for 15 minutes to 1 hour. In other embodiments, at least 5%-10%, atleast 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, atleast 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, orat least 90%-100% of the peptide remains intact after exposure to 100°C. for 15 minutes to 1 hour.

In some embodiments, the peptide of the peptide active agent conjugatecomprises a sequence that has at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 97%, at least 99% or 100% sequenceidentity with any one of SEQ ID NO: 508—SEQ ID NO: 758 or a fragmentthereof. In some embodiments, the peptide of the peptide active agentconjugate comprises a sequence that has at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 99% or100% sequence identity with any one of SEQ ID NO: 744—SEQ ID NO: 758 ora fragment thereof. In some embodiments, the peptide of the peptideactive agent conjugate comprises a sequence that has at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 99% or 100% sequence identity with any one of SEQ ID NO: 798—SEQID NO: 1048 or a fragment thereof. In some embodiments, the peptide ofthe peptide active agent conjugate comprises a sequence that has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99% or 100% sequence identity with any one of SEQ IDNO: 1034—SEQ ID NO: 1048 or a fragment thereof. In some embodiments, thepeptide of the peptide active agent conjugate comprises a sequence ofany one of SEQ ID NO: 508—SEQ ID NO: 758 or a fragment thereof. In someembodiments, the peptide of the peptide active agent conjugate comprisesa sequence of any one of SEQ ID NO: 744—SEQ ID NO: 758 or a fragmentthereof. In some embodiments, the peptide of the peptide active agentconjugate comprises a sequence of any one of SEQ ID NO: 798—SEQ ID NO:1048 or a fragment thereof. In some embodiments, the peptide of thepeptide active agent conjugate comprises a sequence of any one of SEQ IDNO: 1034—SEQ ID NO: 1048 or a fragment thereof. In some embodiments, thepeptide comprises a sequence that has at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 99%, or 100% sequence identity with any one of SEQ ID NO: 744—SEQID NO: 758. In some embodiments, the peptide comprises at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99%, or 100% sequence identity with any one of SEQID NO: 1034-SEQ ID NO: 1048. In some embodiments, the peptide activeagent conjugate or the peptide comprises a sequence of any one of SEQ IDNO: 485—SEQ ID NO: 507 or a fragment thereof. In some embodiments, thepeptide active agent conjugate or the peptide comprises a sequence ofany one of SEQ ID NO: 759—SEQ ID NO: 781 or a fragment thereof. In someembodiments, the peptide active agent conjugate or the peptide comprisesa sequence of any one of SEQ ID NO: 505—SEQ ID NO: 507 or a fragmentthereof. In some embodiments, the peptide active agent conjugate or thepeptide comprises a sequence of any one of SEQ ID NO: 779—SEQ ID NO: 781or a fragment thereof. In some embodiments, the peptide active agentconjugate or the peptide comprises a peptide with at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least, 97%, at least 98%, or at least 99%identical to any one of SEQ ID NO: 978—SEQ ID NO: 1024 or at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 97%, at least 98%, or at least 99%identical to any one of SEQ ID NO: 688—SEQ ID NO: 734.

Pharmacokinetics of Peptides

The pharmacokinetics of any of the peptides of this disclosure can bedetermined after administration of the peptide via different routes ofadministration. For example, the pharmacokinetic parameters of a peptideof this disclosure can be quantified after intravenous, subcutaneous,intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary,transdermal, vaginal, optic, nasal, oral, sublingual, inhalation,dermal, intrathecal, intranasal, intra-articular, peritoneal, buccal,synovial, or topical administration. Peptides of the present disclosurecan be analyzed by using tracking agents such as radiolabels orfluorophores. For example, a radiolabeled peptide of this disclosure canbe administered via various routes of administration. Peptideconcentration or dose recovery in various biological samples such asplasma, urine, feces, any organ, skin, muscle, and other tissues can bedetermined using a range of methods including HPLC, fluorescencedetection techniques (TECAN quantification, flow cytometry, iVIS), orliquid scintillation counting.

The methods and compositions described herein can relate topharmacokinetics of peptide administration via any route to a subject.Pharmacokinetics can be described using methods and models, for example,compartmental models or noncompartmental methods. Compartmental modelsinclude but are not limited to monocompartmental model, the twocompartmental model, the multicompartmental model or the like. Modelscan be divided into different compartments and can be described by thecorresponding scheme. For example, one scheme is the absorption,distribution, metabolism and excretion (ADME) scheme. For anotherexample, another scheme is the liberation, absorption, distribution,metabolism and excretion (LADME) scheme. In some aspects, metabolism andexcretion can be grouped into one compartment referred to as theelimination compartment. For example, liberation can include liberationof the active portion of the composition from the delivery system,absorption includes absorption of the active portion of the compositionby the subject, distribution includes distribution of the compositionthrough the blood plasma and to different tissues, metabolism, whichincludes metabolism or inactivation of the composition and finallyexcretion, which includes excretion or elimination of the composition orthe products of metabolism of the composition. Compositions administeredintravenously to a subject can be subject to multiphasic pharmacokineticprofiles, which can include but are not limited to aspects of tissuedistribution and metabolism/excretion. As such, the decrease in plasmaor serum concentration of the composition is often biphasic, including,for example an alpha phase and a beta phase, occasionally a gamma, deltaor other phase is observed

Pharmacokinetics includes determining at least one parameter associatedwith administration of a peptide to a subject. In some aspects,parameters include at least the dose (D), dosing interval (τ), areaunder curve (AUC), maximum concentration (C_(max)), minimumconcentration reached before a subsequent dose is administered(C_(min)), minimum time (T_(min)), maximum time to reach Cmax (T_(max)),volume of distribution (V_(d)), steady-state volume of distribution(V_(ss)), back-extrapolated concentration at time 0 (C₀), steady stateconcentration (C_(ss)), elimination rate constant (k_(e)), infusion rate(k_(in)), clearance (CL), bioavailability (f), fluctuation (% PTF) andelimination half-life (t_(1/2)).

In certain embodiments, the peptides of any of SEQ ID NO: 485—SEQ ID NO:1048 exhibit optimal pharmacokinetic parameters after oraladministration. In other embodiments, the peptides of any of SEQ ID NO:485—SEQ ID NO: 1048 exhibit optimal pharmacokinetic parameters after anyroute of administration, such as oral administration, inhalation,intranasal administration, topical administration, parenteraladministration, intravenous administration, subcutaneous administration,intra-articular administration, intramuscular administration,intraperitoneal administration, transdermal administration, dermaladministration, or any combination thereof.

In some embodiments any peptide of SEQ ID NO: 485—SEQ ID NO: 1048exhibits an average T_(max) of 0.5-12 hours, or 1-48 hours at which theC_(max) is reached, an average bioavailability in serum of 0.1%-10% inthe subject after administering the peptide to the subject by an oralroute, an average bioavailability in serum of less than 0.1% after oraladministration to a subject for delivery to the GI tract, an averagebioavailability in serum of 10-100% after parenteral administration, anaverage t_(1/2) of 0.1 hours-168 hours, or 0.25 hours-48 hours in asubject after administering the peptide to the subject, an averageclearance (CL) of 0.5-100 L/hour or 0.5-50 L/hour of the peptide afteradministering the peptide to a subject, an average volume ofdistribution (V_(d)) of 200-20,000 mL in the subject after systemicallyadministering the peptide to the subject, or optionally no systemicuptake, any combination thereof.

Methods of Manufacture

Various expression vector/host systems can be utilized for theproduction of the recombinant expression of peptides described herein.Non-limiting examples of such systems include microorganisms such asbacteria transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing a nucleic acid sequenceencoding peptides or peptide fusion proteins/chimeric proteins describedherein, yeast transformed with recombinant yeast expression vectorscontaining the aforementioned nucleic acid sequence, insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the aforementioned nucleic acid sequence, plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV), tobacco mosaic virus (TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing theaforementioned nucleic acid sequence, or animal cell systems infectedwith recombinant virus expression vectors (e.g., adenovirus, vacciniavirus) including cell lines engineered to contain multiple copies of theaforementioned nucleic acid sequence, either stably amplified (e.g.,CHO/dhfr, CHO/glutamine synthetase) or unstably amplified indouble-minute chromosomes (e.g., murine cell lines). Disulfide bondformation and folding of the peptide could occur during expression orafter expression or both.

A host cell can be adapted to express one or more peptides describedherein. The host cells can be prokaryotic, eukaryotic, or insect cells.In some cases, host cells are capable of modulating the expression ofthe inserted sequences, or modifying and processing the gene or proteinproduct in the specific fashion desired. For example, expression fromcertain promoters can be elevated in the presence of certain inducers(e.g., zinc and cadmium ions for metallothionine promoters). In somecases, modifications (e.g., phosphorylation) and processing (e.g.,cleavage) of peptide products can be important for the function of thepeptide. Host cells can have characteristic and specific mechanisms forthe post-translational processing and modification of a peptide. In somecases, the host cells used to express the peptides secretes minimalamounts of proteolytic enzymes.

In the case of cell- or viral-based samples, organisms can be treatedprior to purification to preserve and/or release a target polypeptide.In some embodiments, the cells are fixed using a fixing agent. In someembodiments, the cells are lysed. The cellular material can be treatedin a manner that does not disrupt a significant proportion of cells, butwhich removes proteins from the surface of the cellular material, and/orfrom the interstices between cells. For example, cellular material canbe soaked in a liquid buffer or, in the case of plant material, can besubjected to a vacuum, in order to remove proteins located in theintercellular spaces and/or in the plant cell wall. If the cellularmaterial is a microorganism, proteins can be extracted from themicroorganism culture medium. Alternatively, the peptides can be packedin inclusion bodies. The inclusion bodies can further be separated fromthe cellular components in the medium. In some embodiments, the cellsare not disrupted. A cellular or viral peptide that is presented by acell or virus can be used for the attachment and/or purification ofintact cells or viral particles. In addition to recombinant systems,Peptides can also be synthesized in a cell-free system using a varietyof known techniques employed in protein and peptide synthesis.

In some cases, a host cell produces a peptide that has an attachmentpoint for a drug. An attachment point could comprise a lysine residue,an N-terminus, a cysteine residue, a cysteine disulfide bond, or anon-natural amino acid. The peptide could also be producedsynthetically, such as by solid-phase peptide synthesis, orsolution-phase peptide synthesis. The peptide could be folded (formationof disulfide bonds) during synthesis or after synthesis or both. Peptidefragments could be produced synthetically or recombinantly and thenjoined together synthetically, recombinantly, or via an enzyme.

FIG. 37 illustrates a schematic of a method of manufacturing a constructthat expresses a peptide of the disclosure, such as the constructsillustrated in FIG. 36 and as described throughout the disclosure and inSEQ ID NO: 485—SEQ ID NO: 1048 provided herein.

In other aspects, the peptides of the present disclosure can be preparedby conventional solid phase chemical synthesis techniques, for exampleaccording to the Fmoc solid phase peptide synthesis method (“Fmoc solidphase peptide synthesis, a practical approach,” edited by W. C. Chan andP. D. White, Oxford University Press, 2000), Boc solid phase peptidesynthesis, or solution phase peptide synthesis. The disulfide bonds canbe formed after cleavage from the resin, such as by air oxidation or abuffer system with a set pH range such as from 7-10 and can contain aredox system such as glutathione/oxidized glutathione orcysteine/cystine. The disulfide bonds can also be formed by selectiveprotection and deprotection of specific cysteine residues followed byoxidation, or on the resin. The peptide can be purified, such as byreversed-phase chromatography at any one or more steps during theproduction process. The peptide can be isolated by lyophilization andcan be in various salt forms, such as TFA salt or ammonium and acetatesalt.

Pharmaceutical Compositions of Peptides

A pharmaceutical composition of the disclosure can be a combination ofany peptide described herein with other chemical components, such ascarriers, stabilizers, diluents, dispersing agents, suspending agents,thickening agents, antioxidants, solubilizers, buffers, osmolytes,salts, surfactants, amino acids, encapsulating agents, bulking agents,cryoprotectants, and/or excipients. The pharmaceutical compositionfacilitates administration of a peptide described herein to an organism.Pharmaceutical compositions can be administered intherapeutically-effective amounts as pharmaceutical compositions byvarious forms and routes including, for example, intravenous,subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic,pulmonary, transdermal, vaginal, optic, nasal, oral, sublingual,inhalation, dermal, intrathecal, intranasal, intra-articular, andtopical administration. A pharmaceutical composition can be administeredin a local or systemic manner, for example, via injection of the peptidedescribed herein directly into an organ, optionally in a depot.

Parenteral injections can be formulated for bolus injection orcontinuous infusion. The pharmaceutical compositions can be in a formsuitable for parenteral injection as a sterile suspension, solution oremulsion in oily or aqueous vehicles, and can contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofa peptide described herein in water soluble form. Suspensions ofpeptides described herein can be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension canalso contain suitable stabilizers or agents which increase thesolubility and/or reduce the aggregation of such peptides describedherein to allow for the preparation of highly concentrated solutions.Alternatively, the peptides described herein can be lyophilized or inpowder form for re-constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use. In some embodiments, a purified peptideis administered intravenously.

A peptide of the disclosure can be applied directly to an organ, or anorgan tissue or cells, such as brain or brain tissue or cancer cells,during a surgical procedure. The recombinant peptides described hereincan be administered topically and can be formulated into a variety oftopically administrable compositions, such as solutions, suspensions,lotions, gels, pastes, medicated sticks, balms, creams, and ointments.Such pharmaceutical compositions can contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the peptide described hereindescribed herein can be administered in pharmaceutical compositions to asubject suffering from a condition that affects the immune system. Insome embodiments, the subject is a mammal such as a human. Atherapeutically-effective amount can vary widely depending on theseverity of the disease, the age and relative health of the subject, thepotency of the compounds used, and other factors.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions comprising a peptide described herein can bemanufactured, for example, by expressing the peptide in a recombinantsystem, purifying the peptide, lyophilizing the peptide, mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or compression processes. The pharmaceuticalcompositions can include at least one pharmaceutically acceptablecarrier, diluent, or excipient and compounds described herein asfree-base or pharmaceutically-acceptable salt form.

Methods for the preparation of peptides described herein comprising thecompounds described herein include formulating the peptide describedherein with one or more inert, pharmaceutically-acceptable excipients orcarriers to form a solid, semi-solid, or liquid composition. Solidcompositions include, for example, powders, tablets, dispersiblegranules, capsules, cachets, and suppositories. These compositions canalso contain minor amounts of nontoxic, auxiliary substances, such aswetting or emulsifying agents, pH buffering agents, and otherpharmaceutically-acceptable additives.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), each of which is incorporated by reference in itsentirety.

Administration of Pharmaceutical Compositions

A pharmaceutical composition of the disclosure can be a combination ofany peptide described herein with other chemical components, such ascarriers, stabilizers, diluents, dispersing agents, suspending agents,thickening agents, and/or excipients. The pharmaceutical compositionfacilitates administration of a peptide described herein to an organism.Pharmaceutical compositions can be administered intherapeutically-effective amounts as pharmaceutical compositions byvarious forms and routes including, for example, intravenous,subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic,pulmonary, transdermal, vaginal, optic, nasal, oral, inhalation, dermal,intra-articular, intrathecal, intranasal, and topical administration. Apharmaceutical composition can be administered in a local or systemicmanner, for example, via injection of the peptide described hereindirectly into an organ, optionally in a depot.

Parenteral injections can be formulated for bolus injection orcontinuous infusion. The pharmaceutical compositions can be in a formsuitable for parenteral injection as a sterile suspension, solution oremulsion in oily or aqueous vehicles, and can contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofa peptide described herein in water-soluble form. Suspensions ofpeptides described herein can be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension canalso contain suitable stabilizers or agents which increase thesolubility and/or reduce the aggregation of such peptides describedherein to allow for the preparation of highly concentrated solutions.Alternatively, the peptides described herein can be lyophilized or inpowder form for re-constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use. In some embodiments, a purified peptideis administered intravenously. A peptide described herein can beadministered to a subject, home, target, migrates to, is retained by,and/or binds to, or be directed to an organ, e.g., the cartilage.

A peptide of the disclosure can be applied directly to an organ, or anorgan tissue or cells, such as cartilage or cartilage tissue or cells,during a surgical procedure. The recombinant peptides described hereincan be administered topically and can be formulated into a variety oftopically administrable compositions, such as solutions, suspensions,lotions, gels, pastes, medicated sticks, balms, creams, and ointments.Such pharmaceutical compositions can contain solubilizers, stabilizers,tonicity enhancing agents, buffers and preservatives.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the peptide described hereindescribed herein are administered in pharmaceutical compositions to asubject suffering from a condition. In some instances the pharmaceuticalcomposition will affect the physiology of the animal, such as the immunesystem, inflammatory response, or other physiologic affect. In someembodiments, the subject is a mammal such as a human. Atherapeutically-effective amount can vary widely depending on theseverity of the disease, the age and relative health of the subject, thepotency of the compounds used, and other factors.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions comprising a peptide described herein can bemanufactured, for example, by expressing the peptide in a recombinantsystem, purifying the peptide, lyophilizing the peptide, mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or compression processes. The pharmaceuticalcompositions can include at least one pharmaceutically acceptablecarrier, diluent, or excipient and compounds described herein asfree-base or pharmaceutically-acceptable salt form.

Methods for the preparation of peptides described herein comprising thecompounds described herein include formulating the peptide describedherein with one or more inert, pharmaceutically-acceptable excipients orcarriers to form a solid, semi-solid, or liquid composition. Solidcompositions include, for example, powders, tablets, dispersiblegranules, capsules, cachets, and suppositories. These compositions canalso contain minor amounts of nontoxic, auxiliary substances, such aswetting or emulsifying agents, pH buffering agents, and otherpharmaceutically-acceptable additives.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE. , Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., PharmaceuticalDosage Forms, Marcel Decker, New York, N.Y., 1980; and PharmaceuticalDosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams& Wilkins 1999), each of which is incorporated by reference in itsentirety.

Use of Peptide in Imaging and Surgical Methods

The present disclosure generally relates to peptides that home, target,migrate to, are retained by, accumulate in, and/or bind to, or aredirected to specific regions, tissues, structures, or cells within thebody and methods of using such peptides. These peptides have the abilityto contact the cartilage, which makes them useful for a variety ofapplications. In particular, the peptides can have applications insite-specific modulation of biomolecules to which the peptides aredirected to. End uses of such peptides can include, for example,imaging, research, therapeutics, theranostics, pharmaceuticals,chemotherapy, chelation therapy, targeted drug delivery, andradiotherapy. Some uses can include targeted drug delivery and imaging.

In some embodiments, the present disclosure provides a method fordetecting a cancer, cancerous tissue, or tumor tissue, the methodcomprising the steps of contacting a tissue of interest with a peptideof the present disclosure, wherein the peptide is conjugated to adetectable agent and measuring the level of binding of the peptide,wherein an elevated level of binding, relative to normal tissue, isindicative that the tissue is a cancer, cancerous tissue or tumortissue.

In some embodiments, the disclosure provides a method of imaging anorgan or body region or region, tissue or structure of a subject, themethod comprising administrating to the subject the peptide or apharmaceutical composition disclosed herein and imaging the subject. Insome embodiments such imaging is used to detect a condition associatedwith cartilage, or a function of the cartilage. In some cases thecondition is an inflammation, a cancer, a degradation, a growthdisturbance, genetic, a tear or an injury, or another suitablecondition. In some cases the condition is a chondrodystrophy, atraumatic rupture or detachment, pain following surgery in regions ofthe body containing cartilage, costochondritis, herniation,polychondritis, arthritis, osteoarthritis, rheumatoid arthritis,ankylosing spondylitis (AS), Systemic Lupus Erythematosus (SLE or“Lupus”), Psoriatic Arthritis (PsA), gout, achondroplasia, or anothersuitable condition. In some case the condition is associated with acancer or tumor of the cartilage. In some cases the condition is a typeof chondroma or chondrosarcoma, whether metastatic or not, or anothersuitable condition. In some embodiments, such as those associated withcancers, the imaging may be associated with surgical removal of thediseased region, tissue, structure or cell of a subject.

Furthermore, the present disclosure provides methods for intraoperativeimaging and resection of a diseased or inflamed tissue, cancer,cancerous tissue, or tumor tissue using a peptide of the presentdisclosure conjugated with a detectable agent. In some embodiments, thediseased or inflamed tissue, cancer, cancerous tissue, or tumor tissueis detectable by fluorescence imaging that allows for intraoperativevisualization of the cancer, cancerous tissue, or tumor tissue using apeptide of the present disclosure. In some embodiments, the peptide ofthe present disclosure is conjugated to one or more detectable agents.In a further embodiment, the detectable agent comprises a fluorescentmoiety coupled to the peptide. In another embodiment, the detectableagent comprises a radionuclide. In some embodiments, imaging is achievedduring open surgery. In further embodiments, imaging is accomplishedusing endoscopy or other non-invasive surgical techniques.

Treatment of Cartilage Disorders

The term “effective amount,” as used herein, can refer to a sufficientamount of an agent or a compound being administered which will relieveto some extent one or more of the symptoms of the disease or conditionbeing treated. The result can be reduction and/or alleviation of thesigns, symptoms, or causes of a disease, or any other desired alterationof a biological system. Compositions containing such agents or compoundscan be administered for prophylactic, enhancing, and/or therapeutictreatments. An appropriate “effective” amount in any individual case canbe determined using techniques, such as a dose escalation study.

The methods, compositions, and kits of this disclosure can comprise amethod to prevent, treat, arrest, reverse, or ameliorate the symptoms ofa condition. The treatment can comprise treating a subject (e.g., anindividual, a domestic animal, a wild animal or a lab animal afflictedwith a disease or condition) with a peptide of the disclosure. Intreating a disease, the peptide can contact the cartilage of a subject.The subject can be a human. A subject can be a human; a non-humanprimate such as a chimpanzee, or other ape or monkey species; a farmanimal such as a cattle, horse, sheep, goat, swine; a domestic animalsuch as a rabbit, dog, and cat; a laboratory animal including a rodent,such as a rat, mouse and guinea pig, or the like. A subject can be ofany age. A subject can be, for example, an elderly adult, adult,adolescent, pre-adolescent, child, toddler, infant, or fetus in utero.

Treatment can be provided to the subject before clinical onset ofdisease. Treatment can be provided to the subject after clinical onsetof disease. Treatment can be provided to the subject after 1 day, 1week, 6 months, 12 months, or 2 years or more after clinical onset ofthe disease. Treatment may be provided to the subject for more than 1day, 1 week, 1 month, 6 months, 12 months, 2 years or more afterclinical onset of disease. Treatment may be provided to the subject forless than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years afterclinical onset of the disease. Treatment can also include treating ahuman in a clinical trial. A treatment can comprise administering to asubject a pharmaceutical composition, such as one or more of thepharmaceutical compositions described throughout the disclosure. Atreatment can comprise a once daily dosing. A treatment can comprisedelivering a peptide of the disclosure to a subject, eitherparenterally, intravenously, subcutaneously, intramuscularly, byinhalation, dermally, intra-articular injection, orally, intrathecally,transdermally, intranasally, via a peritoneal route, or directly onto orinto a joint, e.g., via topical, intra-articular injection route orinjection route of application. A treatment can comprise administering apeptide-active agent complex to a subject, either parenterally,intravenously, subcutaneously, intramuscularly, by inhalation, dermally,intra-articular injection, orally, intrathecally, transdermally,intranasally, via a peritoneal route, or directly onto or into a jointor directly onto, near or into the cartilage, e.g., via topical,intra-articular injection route or injection route of application.

Types of cartilage diseases or conditions that can be treated with apeptide of the disclosure can include inflammation, pain management,anti-infective, pain relief, anti-cytokine, cancer, injury, degradation,genetic basis, remodeling, hyperplasia, surgical injury/trauma, or thelike. Diseases or conditions of bone adjacent to cartilage can also betreated with a peptide of the disclosure. Examples of cartilage diseasesor conditions that can be treated with a peptide of the disclosureinclude Costochondritis, Spinal disc herniation, Relapsingpolychondritis, Injury to the articular cartilage, any manner ofrheumatic disease (e.g., Rheumatoid Arthritis (RA), ankylosingspondylitis (AS), Systemic Lupus Erythematosus (SLE or “Lupus”),Psoriatic Arthritis (PsA), Osteoarthritis, Gout, and the like),Herniation, Achondroplasia, Benign or non-cancerous chondroma, Malignantor cancerous chondrosarcoma, Chondriodystrophies, Chondromalaciapatella, Costochondritis, Halus rigidus, Hip labral tear,Osteochondritis dssecans, Osteochondrodysplasias, Torn meniscus, Pectuscarinatum, Pectus excavatum, Chondropathy, Chondromalacia,Polychondritis, Relapsing Polychondritis, Slipped epiphysis,Osteochondritis Dissecans, Chondrodysplasia, Costochondritis,Perichondritis, Osteochondroma, Knee osteoarthritis, Fingerosteoarthritis, Wrist osteoarthritis, Hip osteoarthritis, Spineosteoarthritis, Chondromalacia, Osteoarthritis Susceptibility, AnkleOsteoarthritis, Spondylosis, Secondary chondrosarcoma, Small andunstable nodules as seen in osteoarthritis, Osteochondroses, Primarychondrosarcoma, Cartilage disorders, scleroderma, collagen disorders,Chondrodysplasia, Tietze syndrome, Dermochondrocorneal dystrophy ofFrancois, Epiphyseal dysplasia multiple 1, Epiphyseal dysplasia multiple2, Epiphyseal dysplasia multiple 3, Epiphyseal dysplasia multiple 4,Epiphyseal dysplasia multiple 5, Ossified Ear cartilages with Mentaldeficiency, Muscle Wasting and Bony Changes, Periosteal chondrosarcoma,Carpotarsal osteochondromatosis, Achondroplasia, Genochondromatosis II,Genochondromatosis, Chondrodysplasia—disorder of sex development,Chondroma, Chordoma, Atelosteogenesis, type 1, Atelosteogenesis TypeIII, Atelosteogenesis, type 2, Pyknoachondrogenesis, Osteoarthropathy offingers familial, Dyschondrosteosis—nephritis, Coloboma of Alar-nasalcartilages with telecanthus, Alar cartilageshypoplasia—coloboma—telecanthus, Pierre Robin syndrome—fetalchondrodysplasia, Dysspondyloenchondromatosis, Achondroplasiaregional—dysplasia abdominal muscle, Osteochondritis Dissecans, FamilialArticular Chondrocalcinosis, Tracheobronchomalacia, Chondritis,Dyschondrosteosis, Jequier-Kozlowski-skeletal dysplasia,Chondrodystrophy, Cranio osteoarthropathy, Tietze's syndrome, Hipdysplasia—ecchondromata, Bessel-Hagen disease, Chondromatosis (benign),Enchondromatosis (benign), Chondrocalcinosis due to apatite crystaldeposition, Meyenburg-Altherr-Uehlinger syndrome,Enchondromatosis—dwarfism—deafness, premature growth plate closure(e.g., due to dwarfism, injury, therapy such as retinoid therapy foradolescent acne, or ACL repair), Astley-Kendall syndrome, Synovialosteochondromatosis, Severe achondroplasia with developmental delay andacanthosis nigricans, Chondrocalcinosis, Stanescu syndrome, Familialosteochondritis dissecans, Achondrogenesis type 1A, Achondrogenesis type2, Achondrogenesis, Langer-Saldino Type, Achondrogenesis type 1B,Achondrogenesis type 1A and 1B, Type IIAchondrogenesis-Hypochondrogenesis, Achondrogenesis, Achondrogenesistype 3, Achondrogenesis type 4, Chondrocalcinosis 1, Chondrocalcinosis2, Chondrocalcinosis familial articular, Diastrophic dysplasia,Fibrochondrogenesis, Hypochondroplasia, Keutel syndrome, MaffucciSyndrome, Osteoarthritis Susceptibility 6, Osteoarthritis Susceptibility5, Osteoarthritis Susceptibility 4, Osteoarthritis Susceptibility 3,Osteoarthritis Susceptibility 2, Osteoarthritis Susceptibility 1,Pseudoachondroplasia, Cauliflower ear, Costochondritis, Growth platefractures, Pectus excavatum, septic arthritis, gout, pseudogout (calciumpyrophosphate deposition disease or CPPD), gouty arthritis, bacterial,viral, or fungal infections in or near the joint, bursitis, tendinitis,arthropathies, or a joint disease condition. Examples of bone diseasesor conditions that can be treated with a peptide of the disclosureinclude osteopenia, post-menopausal bone loss, bone maintenance, bonefracture, arthroplasty recovery, osteoporosis, bone loss due tometastatic cancer, fractures due to bone loss (e.g., hip fractures inpatients with osteoporosis), pathological fracture, or atypicalfracture.

In some embodiments, a peptide or peptide conjugate of this disclosurecan be administered to a subject in order to target, an arthritic joint.In other embodiments, a peptide or peptide conjugate of this disclosurecan be administered to a subject in order to treat an arthritic joint.

In some embodiments, the present disclosure provides a method fortreating a cancer, the method comprising administering to a subject inneed thereof an effective amount of a peptide of the present disclosure.

In some embodiments, the present disclosure provides a method fortreating a cancer, the method comprising administering to a patient inneed thereof an effective amount of a pharmaceutical compositioncomprising a peptide of the present disclosure and a pharmaceuticallyacceptable carrier.

In some embodiments, the peptides of the present disclosure can be usedto treat chondrosarcoma. Chondrosarcoma is a cancer of cartilageproducing cells and is often found in bones and joints. It falls withinthe family of bone and soft-tissue sarcomas. In certain embodiments,administration of a peptide or peptide conjugate of the presentdisclosure can be used to image and diagnose or target and treat asubject with chondrosarcoma. The administration of a peptide or peptideconjugate of the present disclosure can be used in combination withablative radiotherapy or proton therapy to treat chondrosarcoma. Thesubject can be a human or an animal.

In some embodiments, a peptide or peptide conjugate of this disclosurecan be used to treat Chordoma. In certain embodiments, administration ofa peptide or peptide conjugate of the present disclosure can be used toimage and diagnose or target and treat a subject with chordoma. Theadministration of a peptide or peptide conjugate of the presentdisclosure can be used in combination with a tyrosine kinase inhibitor,such as imatinib mesylate, and ablative radiotherapy or proton therapyto treat chordoma. The administration of a peptide or peptide conjugateof the present disclosure can be used in combination with anantivascular agent such as bevacizumab and an epidermal growth factorreceptor inhibitor such as erlotinib to treat chordoma. The subject canbe a human or an animal.

In some embodiments, the present disclosure provides a method forinhibiting invasive activity of cells, the method comprisingadministering an effective amount of a peptide of the present disclosureto a subject.

In some embodiments, the peptides of the present disclosure areconjugated to one or more therapeutic agents. In further embodiments,the therapeutic agent is a chemotherapeutic, anti-cancer drug, oranti-cancer agent selected from, but are not limited to:anti-inflammatories, such as for example a glucocorticoid, acorticosteroid, a protease inhibitor, such as for example collagenaseinhibitor or a matrix metalloprotease inhibitor (i.e., MMP-13inhibitor), an amino sugar, vitamin (e.g., Vitamin D), and antibiotics,antiviral, or antifungal, a statin, an immune modulator, radioisotopes,toxins, enzymes, sensitizing drugs, nucleic acids, including interferingRNAs, antibodies, anti-angiogenic agents, cisplatin, anti-metabolites,mitotic inhibitors, growth factor inhibitors, paclitaxel, temozolomide,topotecan, fluorouracil, vincristine, vinblastine, procarbazine,decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine,fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine,etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin,dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen,flutamide, leuprolide, goserelin, aminogluthimide, anastrozole,amsacrine, asparaginase, mitoxantrone, mitotane and amifostine, andtheir equivalents, as well as photo-ablation. Some of these activeagents induce programmed cell death such as apoptosis in target cellsand thereby improve symptoms or ameliorate disease. Apoptosis can beinduced by many active agents, including, for example,chemotherapeutics, anti-inflammatories, corticosteroids, NSAIDS, tumornecrosis factor alpha (TNF-α) modulators, tumor necrosis factor receptor(TNFR) family modulators. In some embodiments, peptides of thisdisclosure can be used to target active agents to pathways of cell deathor cell killing, such as caspases, apoptosis activators and inhibitors,XBP-1, Bc1-2, Bc1-Xl, Bcl-w, and other disclosed herein. In otherembodiments, the therapeutic agent is any nonsteroidal anti-inflammatorydrug (NSAID). The NSAID can be any heterocyclic acetic acid derivativessuch as ketorolac, indomethacin, etodolac, or tolemetin, any propionicacid derivatives such as naproxen, any enolic acid derivatives, anyanthranilic acid derivatives, any selective COX-2 inhibitors such ascelecoxib, any sulfonanilides, any salicylates, aceclofenac, nabumetone,sulindac, diclofenac, or ibuprofen. In other embodiments, thetherapeutic agent is any steroid, such as dexamethasone, budesonide,beclomethasone monopropionate, desciclesonide, triamcinolone, cortisone,prednisone, prednisolone, triamcinolone hexacetonide, ormethylprednisolone. In other embodiments, the therapeutic agent is apain reliever, such as acetaminophen, opioids, local anesthetics,anti-depressants, glutamate receptor antagonists, adenosine, orneuropeptides. In some embodiments, a treatment consists ofadministering a combination of any of the above therapeutic agents and apeptide conjugate, such as a treatment in which both adexamethasone-peptide conjugate and an NSAID are administered to apatient. Peptides of the current disclosure that target the cartilagecan be used to treat the diseases conditions as described herein, forexample, any diseases or conditions including tears, injuries (i.e.,sports injuries), genetic factors, degradation, thinning, inflammation,cancer or any other disease or condition of the cartilage or to targettherapeutically-active substances to treat these diseases amongstothers. In other cases, a peptide of the disclosure can be used to treattraumatic rupture, detachment, chostochondritis, spinal disc herniation,relapsing and non-relapsing polychondritis, injury to the articularcartilage, osteoarthritis, arthritis or achondroplasia. In some cases,the peptide or peptide-active agent can be used to target cancer in thecartilage, for example benign chondroma or malignant chondrosarcoma, bycontacting the cartilage by diffusion into chondrocytes and then havingantitumor function, targeted toxicity, inhibiting metastases, etc. Aswell, such peptide or peptide-active agent can be used to label, detect,or image such cartilage lesions, including tumors and metastases amongstother lesions, which may be removed through various surgical techniquesor by targeting with peptide-active agents that induce programmed celldeath or kill cells.

Venom or toxin derived peptide(s), peptides, modified peptides, labeledpeptides, peptide-active agent conjugates and pharmaceuticalcompositions described herein can be administered for prophylacticand/or therapeutic treatments. In therapeutic applications, thecomposition can be administered to a subject already suffering from adisease or condition, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease or condition, or to cure,heal, improve, or ameliorate the condition. Such peptides describedherein can also be administered to prevent (either in whole or in part),lessen a likelihood of developing, contracting, or worsening acondition. Amounts effective for this use can vary based on the severityand course of the disease or condition, previous therapy, the subject'shealth status, weight, response to the drugs, and the judgment of thetreating physician. Venom or toxin derived peptide(s), peptides,modified peptides, labeled peptides, peptide-active agent conjugates andpharmaceutical compositions described herein can allow for targetedhoming of the peptide and local delivery of any conjugate. For example,a peptide conjugated to a steroid allows for local delivery of thesteroid, which is significantly more effective and less toxic thantraditional systemic steroids. A peptide conjugated to an NSAID isanother example. In this case, the peptide conjugated to an NSAID allowsfor local delivery of the NSAID, which allows for administration of alower NSAID dose and is subsequently less toxic. By delivering an activeagent to the joint, pain relief can be more rapid, may be more longlasting, and can be obtained with a lower systemic dose and off-siteundesired effects than with systemic dosing without targeting.

Peptides of the current disclosure that target the cartilage can be usedto treat or manage pain associated with a cartilage injury or disorder,or any other cartilage or joint condition as described herein. Thepeptides can be used either directly or as carriers of active drugs,peptides, or molecules. For example, since ion channels can beassociated with pain and can be activated in disease states such asarthritis, peptides that interact with ion channels can be used directlyto reduce pain. In another embodiment, the peptide is conjugated to anactive agent with anti-inflammatory activity, in which the peptide actsas a carrier for the local delivery of the active agent to reduce pain.

In some embodiments, the peptides described herein provide a method oftreating a cartilage condition of a subject, the method comprisingadministering to the subject a therapeutically-effective amount of apeptide comprising the sequence SEQ ID NO: 485 or fragment thereof. Insome embodiments, the peptides described herein provide a method oftreating a cartilage condition of a subject, the method comprisingadministering to the subject a peptide of any one of SEQ ID NO: 486—SEQID NO: 1048 or fragment thereof.

Treatment of Kidney Disorders

In some embodiments, peptides of this disclosure that home, target, aredirected to, migrate to, are retained by, accumulate in, or bind tospecific regions, tissues, structures or cells of the kidneys can beused to treat a kidney disorder. In other embodiments, peptides are usedin peptide conjugates of the present disclosure to deliver an activeagent for treatment of a kidney disorder.

In some embodiments, the peptides and peptide-conjugates of the presentdisclosure are used to treat a condition of the kidney, or a region,tissue, structure, or cell thereof. In certain embodiments, thecondition is associated with kidney, or a function of a subject'skidneys. The present disclosure encompasses various acute and chronicrenal diseases, including glomerular, tubule-interstitial, andmicrovascular diseases. Examples of conditions applicable to the presentdisclosure include but are not limited to: hypertensive kidney damage,acute kidney diseases and disorders (AKD), acute kidney injury (AKI) dueto ischemia-reperfusion injury, drug treatment such as chemotherapy,cardiovascular surgery, surgery, medical interventions or treatment,radiocontrast nephropathy, or induced by cisplatin or carboplatin, whichcan be treated prophylactically, established AKI including ischemicrenal injury, endotoxemia-induced AKI, endotoxemia/sepsis syndrome, orestablished nephrotoxic AKI (e.g., rhabdomyolysis, radiocontrastnephropathy, cisplatin/carboplatin AKI, aminoglycoside nephrotoxicity),end stage renal disease, acute and rapidly progressiveglomerulonephritis, acute presentations of nephrotic syndrome, acutepyelonephritis, acute renal failure, chronic glomerulonephritis, chronicheart failure, chronic interstitial nephritis, graft versus host diseaseafter renal transplant, chronic kidney disease (CKD) such as diabeticnephropathy, hypertensive nephrosclerosis, idiopathic chronicglomerulonephritis (e.g., focal glomerular sclerosis, membranousnephropathy, membranoproliferative glomerulonephritis, minimal changedisease transition to chronic disease, anti-GBM disease, rapidlyprogressive cresentic glomerulonephritis, IgA nephropathy), secondarychronic glomerulonephritis (e.g., systemic lupus, polyarteritis nodosa,scleroderma, amyloidosis, endocarditis), hereditary nephropathy (e.g.,polycystic kidney disease, Alport's syndrome), interstitial nephritisinduced by drugs (e.g., Chinese herbs, NSAIDs), multiple myeloma orsarcoid, or renal transplantation such as donor kidney prophylaxis(treatment of donor kidney prior to transplantation), treatment posttransplantation to treat delayed graft function, acute rejection, orchronic rejection, chronic liver disease, chronic pyelonephritis,diabetes, diabetic kidney disease, fibrosis, focal segmentalglomerulosclerosis, Goodpasture's disease, hypertensive nephrosclerosis,IgG4-related renal disease, interstitial inflammation, lupus nephritis,nephritic syndrome, partial obstruction of the urinary tract, polycystickidney disease, progressive renal disease, renal cell carcinoma, renalfibrosis, and vasculitis. For example, in certain embodiments, thepeptides and peptide-conjugates of the present disclosure are used toreduce acute kidney injury in order to prevent it from progressing tochronic kidney disease.

Alternatively or in combination, in some embodiments, the peptide andpeptide-conjugates of the present disclosure are used to elicit aprotective response such as ischemic preconditioning and/or acquiredcytoresistance in a kidney of the subject. In some embodiments, ischemicpreconditioning and/or acquired cytoresistance is induced byadministering an agent (e.g., a peptide or peptide-conjugate of thepresent disclosure) that upregulates the expression of protective stressproteins, such as antioxidants, anti-inflammatory proteins, or proteaseinhibitors. In certain embodiments, the induced response protects thekidney by preserving kidney function in whole or in part and/or byreducing injury to renal tissues and cells, e.g., relative to thesituation where no protective response is induced. The peptides andpeptide-conjugates of the present disclosure can provide certainbenefits compared to other agents for inducing ischemic preconditioningand/or acquired cytoresistance, such as a well-defined chemicalstructure and avoidance of low pH precipitation.

In some embodiments, the protective response is induced in order toprotect the kidney or tissues or cells thereof from an injury or insultthat is predicted to occur (e.g., associated with a planned event suchas a medical procedure, is likely to occur due to a condition in thesubject) or has already occurred. In certain embodiments, the inducedresponse prevents or reduces the extent of damage to the kidney ortissues or cells thereof caused by the injury or insult. For instance,in certain embodiments, the peptides and peptide-conjugates induceacquired cytoresistance by activating protective pathways and/orupregulating expression of protective stress proteins. Optionally, thepeptides and peptide-conjugates are capable of inducing such protectiveresponses while causing minimal or no injury to the kidney.

In various embodiments, the injury or insult is associated with one ormore of: surgery, radiocontrast imaging, cardiopulmonary bypass, balloonangioplasty, induced cardiac or cerebral ischemic-reperfusion injury,organ transplantation, sepsis, shock, low blood pressure, high bloodpressure, kidney hypoperfusion, chemotherapy, drug administration,nephrotoxic drug administration, blunt force trauma, puncture, poison,or smoking. For instance, in certain embodiments, the injury or insultis associated with a medical procedure that has been or will beperformed on the subject, such as one or more of: surgery, radiocontrastimaging, cardiopulmonary bypass, balloon angioplasty, induced cardiac orcerebral ischemic-reperfusion injury, organ transplantation,chemotherapy, drug administration, or nephrotoxic drug administration.

In some embodiments, the peptide itself exhibits a renal therapeuticeffect. For example, in certain embodiments, the cystine-dense peptideinteracts with a renal ion channel, inhibits a protease, hasantimicrobial activity, has anticancer activity, has anti-inflammatoryactivity, induces ischemic preconditioning or acquired cytoresistance,or produces a protective or therapeutic effect on a kidney of thesubject, or a combination thereof. Optionally, the renal therapeuticeffect exhibited by the peptide is a renal protective effect or renalprophylactic effect (e.g., ischemic preconditioning or acquiredcytoresistance) that protects the kidney or a tissue or cell thereoffrom an upcoming injury or insult.

For example, in certain embodiments, a peptide of the present disclosureactivates protective pathways and/or upregulates expression ofprotective stress proteins in the kidney or tissues or cells thereof. Asanother example, in certain embodiments, a peptide of the presentdisclosure accesses and suppresses intracellular injury pathways. In yetanother example, in certain embodiments, a peptide of the presentdisclosure inhibits interstitial inflammation and prevents renalfibrosis. As a further example, in certain embodiments, a peptide of thepresent disclosure is administered prior to or currently with theadministration of a nephrotoxic agent (e.g., aminoglycoside antibioticssuch as gentamicin and minocycline, chemotherapeutics such as cisplatin,immunoglobulins or fragments thereof, mannitol, NSAIDs such as ketorolacor ibuprofen, cyclosporin, cyclophosphamide, radiocontrast dyes) inorder to minimize its damaging effects, e.g., by blockingmegalin-cubulin binding sites so that the nephrotoxic agent passesthrough the kidneys.

In some embodiments, the present disclosure provides that any peptide ofthe disclosure including SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048 can as a peptide conjugate with an active agent fortreatment of a kidney disorder. For example, a peptide of SEQ ID NO:511, SEQ ID NO: 592, or SEQ ID NO: 683 can be conjugated to an activeagent and administered to a subject in need thereof to treat a kidneydisorder.

In some embodiments, homing of a peptide of this disclosure to cartilageor the kidneys can be assessed in an animal model such as thosedescribed in Alves et al. (Clin Rev Allergy Immunol. 2016 August;51(1):27-47. doi: 10. 1007/s12016-015-8522-7), Kuyinu et al. (J OrthopSurg Res. 2016 Feb. 2; 11:19. doi: 10. 1186/s13018-016-0346-5), Li etal. (Exp Biol Med (Maywood). 2015 August; 240(8):1029-38. doi: 10.1177/1535370215594583), and Mullins et al. (Dis Model Mech. 2016 Dec. 1;9(12):1419-1433), all of which are incorporated herein by reference.

Multiple peptides described herein can be administered in any order orsimultaneously. In some cases, multiple functional fragments of peptidesderived from toxins or venom can be administered in any order orsimultaneously. If simultaneously, the multiple peptides describedherein can be provided in a single, unified form, such as an intravenousinjection, or in multiple forms, such as subsequent intravenous dosages.

Peptides can be packaged as a kit. In some embodiments, a kit includeswritten instructions on the use or administration of the peptides.

Conjugate Compounds

The present disclosure provides compounds that selectively bind tocancerous cells and tissues. In various aspects, the compounds of thepresent disclosure comprise a peptide portion and a detectable agentconjugated together.

In various aspects of the present disclosure, the peptide portions ofthe compounds described herein have certain features in common with thenative chlorotoxin (CTX) peptide. The native chlorotoxin peptide wasoriginally isolated from the scorpion Leiurus quinquestriatus.Chlorotoxin is a 36 amino acid peptide that selectively binds tocancerous cells. The peptide portions of the present compounds haveadvantageously retained at least some of the cancer-cell bindingactivity of chlorotoxin. The cancer-cell binding activity of chlorotoxinprovides certain advantages for the detection and treatment of cancerbecause it facilitates the selective localization of detectable agentsand therapeutic agents to the cancer cells for the detection andtreatment of cancer.

Table 1 below sets forth certain polypeptide sequences for use with thepresent disclosure. Citrulline is designated as “Cit” in the sequences.

TABLE 1  Exemplary peptide sequences suitable for use in  the compounds of the present disclosure. SEQ ID NO. Polypeptide Sequence  1 MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR   2MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR   3MCMPCFTTDHQMARRCDDCCGGKGRGKCYGPQCLCR   4MCMPCFTTDHQMARKCDDCCGGAGRGKCYGPQCLCR   5MCMPCFTTDHQMARACDDCCGGAGRGKCYGPQCLCR   6MCMPCFTTDHQMARRCDDCCGGAGRGKCYGPQCLCR   7MCMPCFTTDHQMARKCDDCCGGRGRGKCYGPQCLCR   8MCMPCFTTDHQMARACDDCCGGRGRGKCYGPQCLCR   9MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR  10MCMPCFTTDHQMARKCDDCCGGKGRGACYGPQCLCR  11MCMPCFTTDHQMARACDDCCGGKGRGACYGPQCLCR  12MCMPCFTTDHQMARRCDDCCGGKGRGACYGPQCLCR  13MCMPCFTTDHQMARKCDDCCGGAGRGACYGPQCLCR  14MCMPCFTTDHQMARACDDCCGGAGRGACYGPQCLCR  15MCMPCFTTDHQMARRCDDCCGGAGRGACYGPQCLCR  16MCMPCFTTDHQMARKCDDCCGGRGRGACYGPQCLCR  17MCMPCFTTDHQMARACDDCCGGRGRGACYGPQCLCR  18MCMPCFTTDHQMARRCDDCCGGRGRGACYGPQCLCR  19MCMPCFTTDHQMARKCDDCCGGKGRGRCYGPQCLCR  20MCMPCFTTDHQMARACDDCCGGKGRGRCYGPQCLCR  21MCMPCFTTDHQMARRCDDCCGGKGRGRCYGPQCLCR  22MCMPCFTTDHQMARKCDDCCGGAGRGRCYGPQCLCR  23MCMPCFTTDHQMARACDDCCGGAGRGRCYGPQCLCR  24MCMPCFTTDHQMARRCDDCCGGAGRGRCYGPQCLCR  25MCMPCFTTDHQMARKCDDCCGGRGRGRCYGPQCLCR  26MCMPCFTTDHQMARACDDCCGGRGRGRCYGPQCLCR  27MCMPCFTTDHQMARRCDDCCGGRGRGRCYGPQCLCR  28MCMPCFTTDHQMARRCDDCCGGRGRGRCYGPQCLCR  29KCMPCFTTDHQMARRCDDCCGGRGRGRCYGPQCLCR  30ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR  31KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR  32MCMPCFTTDHQMAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR  33MCMPCFTTDHQMAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR  34KCMPCFTTDHQMAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR  35ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR  36ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR  37KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR  38MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCRGAGAAGG  39MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCRGAGAAGG  40MCMPCFTTDHQMARRCDDCCGGKGRGKCYGPQCLCRGAGAAGG  41MCMPCFTTDHQMARKCDDCCGGAGRGKCYGPQCLCRGAGAAGG  42MCMPCFTTDHQMARACDDCCGGAGRGKCYGPQCLCRGAGAAGG  43MCMPCFTTDHQMARRCDDCCGGAGRGKCYGPQCLCRGAGAAGG  44MCMPCFTTDHQMARKCDDCCGGRGRGKCYGPQCLCRGAGAAGG  45MCMPCFTTDHQMARACDDCCGGRGRGKCYGPQCLCRGAGAAGG  46MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCRGAGAAGG  47MCMPCFTTDHQMARKCDDCCGGKGRGACYGPQCLCRGAGAAGG  48MCMPCFTTDHQMARACDDCCGGKGRGACYGPQCLCRGAGAAGG  49MCMPCFTTDHQMARRCDDCCGGKGRGACYGPQCLCRGAGAAGG  50MCMPCFTTDHQMARKCDDCCGGAGRGACYGPQCLCRGAGAAGG  51MCMPCFTTDHQMARACDDCCGGAGRGACYGPQCLCRGAGAAGG  52MCMPCFTTDHQMARRCDDCCGGAGRGACYGPQCLCRGAGAAGG  53MCMPCFTTDHQMARKCDDCCGGRGRGACYGPQCLCRGAGAAGG  54MCMPCFTTDHQMARACDDCCGGRGRGACYGPQCLCRGAGAAGG  55MCMPCFTTDHQMARRCDDCCGGRGRGACYGPQCLCRGAGAAGG  56MCMPCFTTDHQMARKCDDCCGGKGRGRCYGPQCLCRGAGAAGG  57MCMPCFTTDHQMARACDDCCGGKGRGRCYGPQCLCRGAGAAGG  58MCMPCFTTDHQMARRCDDCCGGKGRGRCYGPQCLCRGAGAAGG  59MCMPCFTTDHQMARKCDDCCGGAGRGRCYGPQCLCRGAGAAGG  60MCMPCFTTDHQMARACDDCCGGAGRGRCYGPQCLCRGAGAAGG  61MCMPCFTTDHQMARRCDDCCGGAGRGRCYGPQCLCRGAGAAGG  62MCMPCFTTDHQMARKCDDCCGGRGRGRCYGPQCLCRGAGAAGG  63MCMPCFTTDHQMARACDDCCGGRGRGRCYGPQCLCRGAGAAGG  64MCMPCFTTDHQMARRCDDCCGGRGRGRCYGPQCLCRGAGAAGG  65MCMPCFTTDHQMARRCDDCCGGRGRGRCYGPQCLCRGAGAAGG  66KCMPCFTTDHQMARRCDDCCGGRGRGRCYGPQCLCRGAGAAGG  67ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCRGAGAAGG  68KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCRGAGAAGG  69MCMPCFTTDHQMAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCRGAGAAGG  70MCMPCFTTDHQMAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG  71KCMPCFTTDHQMAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG  72ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCRGAGAAGG  73ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG  74KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG  75MCMPCFTTDHQMVRKCDDCCGGKGRGKCYGPQCLCR  76MCMPCFTTDHQMVRVCDDCCGGKGRGKCYGPQCLCR  77MCMPCFTTDHQMVRRCDDCCGGKGRGKCYGPQCLCR  78MCMPCFTTDHQMVRKCDDCCGGVGRGKCYGPQCLCR  79MCMPCFTTDHQMVRVCDDCCGGVGRGKCYGPQCLCR  80MCMPCFTTDHQMVRRCDDCCGGVGRGKCYGPQCLCR  81MCMPCFTTDHQMVRKCDDCCGGRGRGKCYGPQCLCR  82MCMPCFTTDHQMVRVCDDCCGGRGRGKCYGPQCLCR  83MCMPCFTTDHQMVRRCDDCCGGRGRGKCYGPQCLCR  84MCMPCFTTDHQMVRKCDDCCGGKGRGVCYGPQCLCR  85MCMPCFTTDHQMVRVCDDCCGGKGRGVCYGPQCLCR  86MCMPCFTTDHQMVRRCDDCCGGKGRGVCYGPQCLCR  87MCMPCFTTDHQMVRKCDDCCGGVGRGVCYGPQCLCR  88MCMPCFTTDHQMVRVCDDCCGGVGRGVCYGPQCLCR  89MCMPCFTTDHQMVRRCDDCCGGVGRGVCYGPQCLCR  90MCMPCFTTDHQMVRKCDDCCGGRGRGVCYGPQCLCR  91MCMPCFTTDHQMVRVCDDCCGGRGRGVCYGPQCLCR  92MCMPCFTTDHQMVRRCDDCCGGRGRGVCYGPQCLCR  93MCMPCFTTDHQMVRKCDDCCGGKGRGRCYGPQCLCR  94MCMPCFTTDHQMVRVCDDCCGGKGRGRCYGPQCLCR  95MCMPCFTTDHQMVRRCDDCCGGKGRGRCYGPQCLCR  96MCMPCFTTDHQMVRKCDDCCGGVGRGRCYGPQCLCR  97MCMPCFTTDHQMVRVCDDCCGGVGRGRCYGPQCLCR  98MCMPCFTTDHQMVRRCDDCCGGVGRGRCYGPQCLCR  99MCMPCFTTDHQMVRKCDDCCGGRGRGRCYGPQCLCR 100MCMPCFTTDHQMVRVCDDCCGGRGRGRCYGPQCLCR 101MCMPCFTTDHQMVRRCDDCCGGRGRGRCYGPQCLCR 102MCMPCFTTDHQMVRRCDDCCGGRGRGRCYGPQCLCR 103KCMPCFTTDHQMVRRCDDCCGGRGRGRCYGPQCLCR 104VCVPCFTTDHQVVRRCDDCCGGRGRGRCYGPQCLCR 105KCVPCFTTDHQVVRRCDDCCGGRGRGRCYGPQCLCR 106MCMPCFTTDHQMVR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 107MCMPCFTTDHQMVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 108KCMPCFTTDHQMVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 109VCVPCFTTDHQVVR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 110VCVPCFTTDHQVVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 111KCVPCFTTDHQVVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 112MCMPCFTTDHQMVRKCDDCCGGKGRGKCYGPQCLCRGAGAAGG 113MCMPCFTTDHQMVRVCDDCCGGKGRGKCYGPQCLCRGAGAAGG 114MCMPCFTTDHQMVRRCDDCCGGKGRGKCYGPQCLCRGAGAAGG 115MCMPCFTTDHQMVRKCDDCCGGVGRGKCYGPQCLCRGAGAAGG 116MCMPCFTTDHQMVRVCDDCCGGVGRGKCYGPQCLCRGAGAAGG 117MCMPCFTTDHQMVRRCDDCCGGVGRGKCYGPQCLCRGAGAAGG 118MCMPCFTTDHQMVRKCDDCCGGRGRGKCYGPQCLCRGAGAAGG 119MCMPCFTTDHQMVRVCDDCCGGRGRGKCYGPQCLCRGAGAAGG 120MCMPCFTTDHQMVRRCDDCCGGRGRGKCYGPQCLCRGAGAAGG 121MCMPCFTTDHQMVRKCDDCCGGKGRGVCYGPQCLCRGAGAAGG 122MCMPCFTTDHQMVRVCDDCCGGKGRGVCYGPQCLCRGAGAAGG 123MCMPCFTTDHQMVRRCDDCCGGKGRGVCYGPQCLCRGAGAAGG 124MCMPCFTTDHQMVRKCDDCCGGVGRGVCYGPQCLCRGAGAAGG 125MCMPCFTTDHQMVRVCDDCCGGVGRGVCYGPQCLCRGAGAAGG 126MCMPCFTTDHQMVRRCDDCCGGVGRGVCYGPQCLCRGAGAAGG 127MCMPCFTTDHQMVRKCDDCCGGRGRGVCYGPQCLCRGAGAAGG 128MCMPCFTTDHQMVRVCDDCCGGRGRGVCYGPQCLCRGAGAAGG 129MCMPCFTTDHQMVRRCDDCCGGRGRGVCYGPQCLCRGAGAAGG 130MCMPCFTTDHQMVRKCDDCCGGKGRGRCYGPQCLCRGAGAAGG 131MCMPCFTTDHQMVRVCDDCCGGKGRGRCYGPQCLCRGAGAAGG 132MCMPCFTTDHQMVRRCDDCCGGKGRGRCYGPQCLCRGAGAAGG 133MCMPCFTTDHQMVRKCDDCCGGVGRGRCYGPQCLCRGAGAAGG 134MCMPCFTTDHQMVRVCDDCCGGVGRGRCYGPQCLCRGAGAAGG 135MCMPCFTTDHQMVRRCDDCCGGVGRGRCYGPQCLCRGAGAAGG 136MCMPCFTTDHQMVRKCDDCCGGRGRGRCYGPQCLCRGAGAAGG 137MCMPCFTTDHQMVRVCDDCCGGRGRGRCYGPQCLCRGAGAAGG 138MCMPCFTTDHQMVRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 139MCMPCFTTDHQMVRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 140KCMPCFTTDHQMVRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 141VCVPCFTTDHQVVRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 142KCVPCFTTDHQVVRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 143MCMPCFTTDHQMVR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCRGAGAAGG 144MCMPCFTTDHQMVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 145KCMPCFTTDHQMVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 146VCVPCFTTDHQVVR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCRGAGAAGG 147VCVPCFTTDHQVVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 148KCVPCFTTDHQVVR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 149MCMPCFTTDHQMLRKCDDCCGGKGRGKCYGPQCLCR 150MCMPCFTTDHQMLRLCDDCCGGKGRGKCYGPQCLCR 151MCMPCFTTDHQMLRRCDDCCGGKGRGKCYGPQCLCR 152MCMPCFTTDHQMLRKCDDCCGGLGRGKCYGPQCLCR 153MCMPCFTTDHQMLRLCDDCCGGLGRGKCYGPQCLCR 154MCMPCFTTDHQMLRRCDDCCGGLGRGKCYGPQCLCR 155MCMPCFTTDHQMLRKCDDCCGGRGRGKCYGPQCLCR 156MCMPCFTTDHQMLRLCDDCCGGRGRGKCYGPQCLCR 157MCMPCFTTDHQMLRRCDDCCGGRGRGKCYGPQCLCR 158MCMPCFTTDHQMLRKCDDCCGGKGRGLCYGPQCLCR 159MCMPCFTTDHQMLRLCDDCCGGKGRGLCYGPQCLCR 160MCMPCFTTDHQMLRRCDDCCGGKGRGLCYGPQCLCR 161MCMPCFTTDHQMLRKCDDCCGGLGRGLCYGPQCLCR 162MCMPCFTTDHQMLRLCDDCCGGLGRGLCYGPQCLCR 163MCMPCFTTDHQMLRRCDDCCGGLGRGLCYGPQCLCR 164MCMPCFTTDHQMLRKCDDCCGGRGRGLCYGPQCLCR 165MCMPCFTTDHQMLRLCDDCCGGRGRGLCYGPQCLCR 166MCMPCFTTDHQMLRRCDDCCGGRGRGLCYGPQCLCR 167MCMPCFTTDHQMLRKCDDCCGGKGRGRCYGPQCLCR 168MCMPCFTTDHQMLRLCDDCCGGKGRGRCYGPQCLCR 169MCMPCFTTDHQMLRRCDDCCGGKGRGRCYGPQCLCR 170MCMPCFTTDHQMLRKCDDCCGGLGRGRCYGPQCLCR 171MCMPCFTTDHQMLRLCDDCCGGLGRGRCYGPQCLCR 172MCMPCFTTDHQMLRRCDDCCGGLGRGRCYGPQCLCR 173MCMPCFTTDHQMLRKCDDCCGGRGRGRCYGPQCLCR 174MCMPCFTTDHQMLRLCDDCCGGRGRGRCYGPQCLCR 175MCMPCFTTDHQMLRRCDDCCGGRGRGRCYGPQCLCR 176MCMPCFTTDHQMLRRCDDCCGGRGRGRCYGPQCLCR 177KCMPCFTTDHQMLRRCDDCCGGRGRGRCYGPQCLCR 178LCLPCFTTDHQLLRRCDDCCGGRGRGRCYGPQCLCR 179KCLPCFTTDHQLLRRCDDCCGGRGRGRCYGPQCLCR 180MCMPCFTTDHQMLR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 181MCMPCFTTDHQMLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 182KCMPCFTTDHQMLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 183LCLPCFTTDHQLLR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 184LCLPCFTTDHQLLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 185KCLPCFTTDHQLLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 186MCMPCFTTDHQMLRKCDDCCGGKGRGKCYGPQCLCRGAGAAGG 187MCMPCFTTDHQMLRLCDDCCGGKGRGKCYGPQCLCRGAGAAGG 188MCMPCFTTDHQMLRRCDDCCGGKGRGKCYGPQCLCRGAGAAGG 189MCMPCFTTDHQMLRKCDDCCGGLGRGKCYGPQCLCRGAGAAGG 190MCMPCFTTDHQMLRLCDDCCGGLGRGKCYGPQCLCRGAGAAGG 191MCMPCFTTDHQMLRRCDDCCGGLGRGKCYGPQCLCRGAGAAGG 192MCMPCFTTDHQMLRKCDDCCGGRGRGKCYGPQCLCRGAGAAGG 193MCMPCFTTDHQMLRLCDDCCGGRGRGKCYGPQCLCRGAGAAGG 194MCMPCFTTDHQMLRRCDDCCGGRGRGKCYGPQCLCRGAGAAGG 195MCMPCFTTDHQMLRKCDDCCGGKGRGLCYGPQCLCRGAGAAGG 196MCMPCFTTDHQMLRLCDDCCGGKGRGLCYGPQCLCRGAGAAGG 197MCMPCFTTDHQMLRRCDDCCGGKGRGLCYGPQCLCRGAGAAGG 198MCMPCFTTDHQMLRKCDDCCGGLGRGLCYGPQCLCRGAGAAGG 199MCMPCFTTDHQMLRLCDDCCGGLGRGLCYGPQCLCRGAGAAGG 200MCMPCFTTDHQMLRRCDDCCGGLGRGLCYGPQCLCRGAGAAGG 201MCMPCFTTDHQMLRKCDDCCGGRGRGLCYGPQCLCRGAGAAGG 202MCMPCFTTDHQMLRLCDDCCGGRGRGLCYGPQCLCRGAGAAGG 203MCMPCFTTDHQMLRRCDDCCGGRGRGLCYGPQCLCRGAGAAGG 204MCMPCFTTDHQMLRKCDDCCGGKGRGRCYGPQCLCRGAGAAGG 205MCMPCFTTDHQMLRLCDDCCGGKGRGRCYGPQCLCRGAGAAGG 206MCMPCFTTDHQMLRRCDDCCGGKGRGRCYGPQCLCRGAGAAGG 207MCMPCFTTDHQMLRKCDDCCGGLGRGRCYGPQCLCRGAGAAGG 208MCMPCFTTDHQMLRLCDDCCGGLGRGRCYGPQCLCRGAGAAGG 209MCMPCFTTDHQMLRRCDDCCGGLGRGRCYGPQCLCRGAGAAGG 210MCMPCFTTDHQMLRKCDDCCGGRGRGRCYGPQCLCRGAGAAGG 211MCMPCFTTDHQMLRLCDDCCGGRGRGRCYGPQCLCRGAGAAGG 212MCMPCFTTDHQMLRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 213MCMPCFTTDHQMLRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 214KCMPCFTTDHQMLRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 215LCLPCFTTDHQLLRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 216KCLPCFTTDHQLLRRCDDCCGGRGRGRCYGPQCLCRGAGAAGG 217MCMPCFTTDHQMLR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCRGAGAAGG 218MCMPCFTTDHQMLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 219KCMPCFTTDHQMLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 220LCLPCFTTDHQLLR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCRGAGAAGG 221LCLPCFTTDHQLLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 222KCLPCFTTDHQLLR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCRGAGAAGG 223GCGPCFTTDHQGARKCDDCCGGKGRGKCYGPQCLCR 224GCGPCFTTDHQGARACDDCCGGKGRGKCYGPQCLCR 225GCGPCFTTDHQGARRCDDCCGGKGRGKCYGPQCLCR 226GCGPCFTTDHQGARKCDDCCGGAGRGKCYGPQCLCR 227GCGPCFTTDHQGARACDDCCGGAGRGKCYGPQCLCR 228GCGPCFTTDHQGARRCDDCCGGAGRGKCYGPQCLCR 229GCGPCFTTDHQGARKCDDCCGGRGRGKCYGPQCLCR 230GCGPCFTTDHQGARACDDCCGGRGRGKCYGPQCLCR 231GCGPCFTTDHQGARRCDDCCGGRGRGKCYGPQCLCR 232GCGPCFTTDHQGARKCDDCCGGKGRGACYGPQCLCR 233GCGPCFTTDHQGARACDDCCGGKGRGACYGPQCLCR 234GCGPCFTTDHQGARRCDDCCGGKGRGACYGPQCLCR 235GCGPCFTTDHQGARKCDDCCGGAGRGACYGPQCLCR 236GCGPCFTTDHQGARACDDCCGGAGRGACYGPQCLCR 237GCGPCFTTDHQGARRCDDCCGGAGRGACYGPQCLCR 238GCGPCFTTDHQGARKCDDCCGGRGRGACYGPQCLCR 239GCGPCFTTDHQGARACDDCCGGRGRGACYGPQCLCR 240GCGPCFTTDHQGARRCDDCCGGRGRGACYGPQCLCR 241GCGPCFTTDHQGARKCDDCCGGKGRGRCYGPQCLCR 242GCGPCFTTDHQGARACDDCCGGKGRGRCYGPQCLCR 243GCGPCFTTDHQGARRCDDCCGGKGRGRCYGPQCLCR 244GCGPCFTTDHQGARKCDDCCGGAGRGRCYGPQCLCR 245GCGPCFTTDHQGARACDDCCGGAGRGRCYGPQCLCR 246GCGPCFTTDHQGARRCDDCCGGAGRGRCYGPQCLCR 247GCGPCFTTDHQGARKCDDCCGGRGRGRCYGPQCLCR 248GCGPCFTTDHQGARACDDCCGGRGRGRCYGPQCLCR 249GCGPCFTTDHQGARRCDDCCGGRGRGRCYGPQCLCR 250GCGPCFTTDHQGARRCDDCCGGRGRGRCYGPQCLCR 251KCGPCFTTDHQGARRCDDCCGGRGRGRCYGPQCLCR 252ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 253KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 254GCGPCFTTDHQGAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 255GCGPCFTTDHQGAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 256KCGPCFTTDHQGAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 257ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 258ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 259KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 260ACAPCFTTDHQAARKCDDCCGGKGRGKCYGPQCLCR 261ACAPCFTTDHQAARACDDCCGGKGRGKCYGPQCLCR 262ACAPCFTTDHQAARRCDDCCGGKGRGKCYGPQCLCR 263ACAPCFTTDHQAARKCDDCCGGAGRGKCYGPQCLCR 264ACAPCFTTDHQAARACDDCCGGAGRGKCYGPQCLCR 265ACAPCFTTDHQAARRCDDCCGGAGRGKCYGPQCLCR 266ACAPCFTTDHQAARKCDDCCGGRGRGKCYGPQCLCR 267ACAPCFTTDHQAARACDDCCGGRGRGKCYGPQCLCR 268ACAPCFTTDHQAARRCDDCCGGRGRGKCYGPQCLCR 269ACAPCFTTDHQAARKCDDCCGGKGRGACYGPQCLCR 270ACAPCFTTDHQAARACDDCCGGKGRGACYGPQCLCR 271ACAPCFTTDHQAARRCDDCCGGKGRGACYGPQCLCR 272ACAPCFTTDHQAARKCDDCCGGAGRGACYGPQCLCR 273ACAPCFTTDHQAARACDDCCGGAGRGACYGPQCLCR 274ACAPCFTTDHQAARRCDDCCGGAGRGACYGPQCLCR 275ACAPCFTTDHQAARKCDDCCGGRGRGACYGPQCLCR 276ACAPCFTTDHQAARACDDCCGGRGRGACYGPQCLCR 277ACAPCFTTDHQAARRCDDCCGGRGRGACYGPQCLCR 278ACAPCFTTDHQAARKCDDCCGGKGRGRCYGPQCLCR 279ACAPCFTTDHQAARACDDCCGGKGRGRCYGPQCLCR 280ACAPCFTTDHQAARRCDDCCGGKGRGRCYGPQCLCR 281ACAPCFTTDHQAARKCDDCCGGAGRGRCYGPQCLCR 282ACAPCFTTDHQAARACDDCCGGAGRGRCYGPQCLCR 283ACAPCFTTDHQAARRCDDCCGGAGRGRCYGPQCLCR 284ACAPCFTTDHQAARKCDDCCGGRGRGRCYGPQCLCR 285ACAPCFTTDHQAARACDDCCGGRGRGRCYGPQCLCR 286ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 287ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 288KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 289ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 290KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 291ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 292ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 293KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 294ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 295ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 296KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 297ICIPCFTTDHQIARKCDDCCGGKGRGKCYGPQCLCR 298ICIPCFTTDHQIARACDDCCGGKGRGKCYGPQCLCR 299ICIPCFTTDHQIARRCDDCCGGKGRGKCYGPQCLCR 300ICIPCFTTDHQIARKCDDCCGGAGRGKCYGPQCLCR 301ICIPCFTTDHQIARACDDCCGGAGRGKCYGPQCLCR 302ICIPCFTTDHQIARRCDDCCGGAGRGKCYGPQCLCR 303ICIPCFTTDHQIARKCDDCCGGRGRGKCYGPQCLCR 304ICIPCFTTDHQIARACDDCCGGRGRGKCYGPQCLCR 305ICIPCFTTDHQIARRCDDCCGGRGRGKCYGPQCLCR 306ICIPCFTTDHQIARKCDDCCGGKGRGACYGPQCLCR 307ICIPCFTTDHQIARACDDCCGGKGRGACYGPQCLCR 308ICIPCFTTDHQIARRCDDCCGGKGRGACYGPQCLCR 309ICIPCFTTDHQIARKCDDCCGGAGRGACYGPQCLCR 310ICIPCFTTDHQIARACDDCCGGAGRGACYGPQCLCR 311ICIPCFTTDHQIARRCDDCCGGAGRGACYGPQCLCR 312ICIPCFTTDHQIARKCDDCCGGRGRGACYGPQCLCR 313ICIPCFTTDHQIARACDDCCGGRGRGACYGPQCLCR 314ICIPCFTTDHQIARRCDDCCGGRGRGACYGPQCLCR 315ICIPCFTTDHQIARKCDDCCGGKGRGRCYGPQCLCR 316ICIPCFTTDHQIARACDDCCGGKGRGRCYGPQCLCR 317ICIPCFTTDHQIARRCDDCCGGKGRGRCYGPQCLCR 318ICIPCFTTDHQIARKCDDCCGGAGRGRCYGPQCLCR 319ICIPCFTTDHQIARACDDCCGGAGRGRCYGPQCLCR 320ICIPCFTTDHQIARRCDDCCGGAGRGRCYGPQCLCR 321ICIPCFTTDHQIARKCDDCCGGRGRGRCYGPQCLCR 322ICIPCFTTDHQIARACDDCCGGRGRGRCYGPQCLCR 323ICIPCFTTDHQIARRCDDCCGGRGRGRCYGPQCLCR 324ICIPCFTTDHQIARRCDDCCGGRGRGRCYGPQCLCR 325KCIPCFTTDHQIARRCDDCCGGRGRGRCYGPQCLCR 326ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 327KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 328ICIPCFTTDHQIAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 329ICIPCFTTDHQIAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 330KCIPCFTTDHQIAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 331ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 332ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 333KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 334TCTPCFTTDHQTARKCDDCCGGKGRGKCYGPQCLCR 335TCTPCFTTDHQTARACDDCCGGKGRGKCYGPQCLCR 336TCTPCFTTDHQTARRCDDCCGGKGRGKCYGPQCLCR 337TCTPCFTTDHQTARKCDDCCGGAGRGKCYGPQCLCR 338TCTPCFTTDHQTARACDDCCGGAGRGKCYGPQCLCR 339TCTPCFTTDHQTARRCDDCCGGAGRGKCYGPQCLCR 340TCTPCFTTDHQTARKCDDCCGGRGRGKCYGPQCLCR 341TCTPCFTTDHQTARACDDCCGGRGRGKCYGPQCLCR 342TCTPCFTTDHQTARRCDDCCGGRGRGKCYGPQCLCR 343TCTPCFTTDHQTARKCDDCCGGKGRGACYGPQCLCR 344TCTPCFTTDHQTARACDDCCGGKGRGACYGPQCLCR 345TCTPCFTTDHQTARRCDDCCGGKGRGACYGPQCLCR 346TCTPCFTTDHQTARKCDDCCGGAGRGACYGPQCLCR 347TCTPCFTTDHQTARACDDCCGGAGRGACYGPQCLCR 348TCTPCFTTDHQTARRCDDCCGGAGRGACYGPQCLCR 349TCTPCFTTDHQTARKCDDCCGGRGRGACYGPQCLCR 350TCTPCFTTDHQTARACDDCCGGRGRGACYGPQCLCR 351TCTPCFTTDHQTARRCDDCCGGRGRGACYGPQCLCR 352TCTPCFTTDHQTARKCDDCCGGKGRGRCYGPQCLCR 353TCTPCFTTDHQTARACDDCCGGKGRGRCYGPQCLCR 354TCTPCFTTDHQTARRCDDCCGGKGRGRCYGPQCLCR 355TCTPCFTTDHQTARKCDDCCGGAGRGRCYGPQCLCR 356TCTPCFTTDHQTARACDDCCGGAGRGRCYGPQCLCR 357TCTPCFTTDHQTARRCDDCCGGAGRGRCYGPQCLCR 358TCTPCFTTDHQTARKCDDCCGGRGRGRCYGPQCLCR 359TCTPCFTTDHQTARACDDCCGGRGRGRCYGPQCLCR 360TCTPCFTTDHQTARRCDDCCGGRGRGRCYGPQCLCR 361TCTPCFTTDHQTARRCDDCCGGRGRGRCYGPQCLCR 362KCTPCFTTDHQTARRCDDCCGGRGRGRCYGPQCLCR 363ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 364KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 365TCTPCFTTDHQTAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 366TCTPCFTTDHQTAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 367KCTPCFTTDHQTAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 368ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 369ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 370KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 371VCVPCFTTDHQVARKCDDCCGGKGRGKCYGPQCLCR 372VCVPCFTTDHQVARACDDCCGGKGRGKCYGPQCLCR 373VCVPCFTTDHQVARRCDDCCGGKGRGKCYGPQCLCR 374VCVPCFTTDHQVARKCDDCCGGAGRGKCYGPQCLCR 375VCVPCFTTDHQVARACDDCCGGAGRGKCYGPQCLCR 376VCVPCFTTDHQVARRCDDCCGGAGRGKCYGPQCLCR 377VCVPCFTTDHQVARKCDDCCGGRGRGKCYGPQCLCR 378VCVPCFTTDHQVARACDDCCGGRGRGKCYGPQCLCR 379VCVPCFTTDHQVARRCDDCCGGRGRGKCYGPQCLCR 380VCVPCFTTDHQVARKCDDCCGGKGRGACYGPQCLCR 381VCVPCFTTDHQVARACDDCCGGKGRGACYGPQCLCR 382VCVPCFTTDHQVARRCDDCCGGKGRGACYGPQCLCR 383VCVPCFTTDHQVARKCDDCCGGAGRGACYGPQCLCR 384VCVPCFTTDHQVARACDDCCGGAGRGACYGPQCLCR 385VCVPCFTTDHQVARRCDDCCGGAGRGACYGPQCLCR 386VCVPCFTTDHQVARKCDDCCGGRGRGACYGPQCLCR 387VCVPCFTTDHQVARACDDCCGGRGRGACYGPQCLCR 388VCVPCFTTDHQVARRCDDCCGGRGRGACYGPQCLCR 389VCVPCFTTDHQVARKCDDCCGGKGRGRCYGPQCLCR 390VCVPCFTTDHQVARACDDCCGGKGRGRCYGPQCLCR 391VCVPCFTTDHQVARRCDDCCGGKGRGRCYGPQCLCR 392VCVPCFTTDHQVARKCDDCCGGAGRGRCYGPQCLCR 393VCVPCFTTDHQVARACDDCCGGAGRGRCYGPQCLCR 394VCVPCFTTDHQVARRCDDCCGGAGRGRCYGPQCLCR 395VCVPCFTTDHQVARKCDDCCGGRGRGRCYGPQCLCR 396VCVPCFTTDHQVARACDDCCGGRGRGRCYGPQCLCR 397VCVPCFTTDHQVARRCDDCCGGRGRGRCYGPQCLCR 398VCVPCFTTDHQVARRCDDCCGGRGRGRCYGPQCLCR 399KCVPCFTTDHQVARRCDDCCGGRGRGRCYGPQCLCR 400ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 401KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 402VCVPCFTTDHQVAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 403VCVPCFTTDHQVAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 404KCVPCFTTDHQVAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 405ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 406ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 407KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 408LCLPCFTTDHQLARKCDDCCGGKGRGKCYGPQCLCR 409LCLPCFTTDHQLARACDDCCGGKGRGKCYGPQCLCR 410LCLPCFTTDHQLARRCDDCCGGKGRGKCYGPQCLCR 411LCLPCFTTDHQLARKCDDCCGGAGRGKCYGPQCLCR 412LCLPCFTTDHQLARACDDCCGGAGRGKCYGPQCLCR 413LCLPCFTTDHQLARRCDDCCGGAGRGKCYGPQCLCR 414LCLPCFTTDHQLARKCDDCCGGRGRGKCYGPQCLCR 415LCLPCFTTDHQLARACDDCCGGRGRGKCYGPQCLCR 416LCLPCFTTDHQLARRCDDCCGGRGRGKCYGPQCLCR 417LCLPCFTTDHQLARKCDDCCGGKGRGACYGPQCLCR 418LCLPCFTTDHQLARACDDCCGGKGRGACYGPQCLCR 419LCLPCFTTDHQLARRCDDCCGGKGRGACYGPQCLCR 420LCLPCFTTDHQLARKCDDCCGGAGRGACYGPQCLCR 421LCLPCFTTDHQLARACDDCCGGAGRGACYGPQCLCR 422LCLPCFTTDHQLARRCDDCCGGAGRGACYGPQCLCR 423LCLPCFTTDHQLARKCDDCCGGRGRGACYGPQCLCR 424LCLPCFTTDHQLARACDDCCGGRGRGACYGPQCLCR 425LCLPCFTTDHQLARRCDDCCGGRGRGACYGPQCLCR 426LCLPCFTTDHQLARKCDDCCGGKGRGRCYGPQCLCR 427LCLPCFTTDHQLARACDDCCGGKGRGRCYGPQCLCR 428LCLPCFTTDHQLARRCDDCCGGKGRGRCYGPQCLCR 429LCLPCFTTDHQLARKCDDCCGGAGRGRCYGPQCLCR 430LCLPCFTTDHQLARACDDCCGGAGRGRCYGPQCLCR 431LCLPCFTTDHQLARRCDDCCGGAGRGRCYGPQCLCR 432LCLPCFTTDHQLARKCDDCCGGRGRGRCYGPQCLCR 433LCLPCFTTDHQLARACDDCCGGRGRGRCYGPQCLCR 434LCLPCFTTDHQLARRCDDCCGGRGRGRCYGPQCLCR 435LCLPCFTTDHQLARRCDDCCGGRGRGRCYGPQCLCR 436KCLPCFTTDHQLARRCDDCCGGRGRGRCYGPQCLCR 437ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 438KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 439LCLPCFTTDHQLAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 440LCLPCFTTDHQLAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 441KCLPCFTTDHQLAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 442ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 443ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 444KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 445SCSPCFTTDHQSARKCDDCCGGKGRGKCYGPQCLCR 446SCSPCFTTDHQSARACDDCCGGKGRGKCYGPQCLCR 447SCSPCFTTDHQSARRCDDCCGGKGRGKCYGPQCLCR 448SCSPCFTTDHQSARKCDDCCGGAGRGKCYGPQCLCR 449SCSPCFTTDHQSARACDDCCGGAGRGKCYGPQCLCR 450SCSPCFTTDHQSARRCDDCCGGAGRGKCYGPQCLCR 451SCSPCFTTDHQSARKCDDCCGGRGRGKCYGPQCLCR 452SCSPCFTTDHQSARACDDCCGGRGRGKCYGPQCLCR 453SCSPCFTTDHQSARRCDDCCGGRGRGKCYGPQCLCR 454SCSPCFTTDHQSARKCDDCCGGKGRGACYGPQCLCR 455SCSPCFTTDHQSARACDDCCGGKGRGACYGPQCLCR 456SCSPCFTTDHQSARRCDDCCGGKGRGACYGPQCLCR 457SCSPCFTTDHQSARKCDDCCGGAGRGACYGPQCLCR 458SCSPCFTTDHQSARACDDCCGGAGRGACYGPQCLCR 459SCSPCFTTDHQSARRCDDCCGGAGRGACYGPQCLCR 460SCSPCFTTDHQSARKCDDCCGGRGRGACYGPQCLCR 461SCSPCFTTDHQSARACDDCCGGRGRGACYGPQCLCR 462SCSPCFTTDHQSARRCDDCCGGRGRGACYGPQCLCR 463SCSPCFTTDHQSARKCDDCCGGKGRGRCYGPQCLCR 464SCSPCFTTDHQSARACDDCCGGKGRGRCYGPQCLCR 465SCSPCFTTDHQSARRCDDCCGGKGRGRCYGPQCLCR 466SCSPCFTTDHQSARKCDDCCGGAGRGRCYGPQCLCR 467SCSPCFTTDHQSARACDDCCGGAGRGRCYGPQCLCR 468SCSPCFTTDHQSARRCDDCCGGAGRGRCYGPQCLCR 469SCSPCFTTDHQSARKCDDCCGGRGRGRCYGPQCLCR 470SCSPCFTTDHQSARACDDCCGGRGRGRCYGPQCLCR 471SCSPCFTTDHQSARRCDDCCGGRGRGRCYGPQCLCR 472SCSPCFTTDHQSARRCDDCCGGRGRGRCYGPQCLCR 473KCSPCFTTDHQSARRCDDCCGGRGRGRCYGPQCLCR 474ACAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 475KCAPCFTTDHQAARRCDDCCGGRGRGRCYGPQCLCR 476SCSPCFTTDHQSAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 477SCSPCFTTDHQSAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 478KCSPCFTTDHQSAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 479ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRGKCYGPQCLCR 480ACAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR 481KCAPCFTTDHQAAR(Cit)CDDCCGG(Cit)GRG(Cit)CYGPQCLCR Cit = Citrulline.

Chlorotoxin conjugates comprise a chlorotoxin and a labeling agent ordetectable label. In an embodiment, chlorotoxin is a variant comprisingat least 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the naturalpeptide of chlorotoxin. In another embodiment, the present disclosureprovides a chlorotoxin having the following amino acid sequence:

-   MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR. In a further embodiment, the    present disclosure provides chlorotoxin variants comprising at least    60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to the following amino acid    sequence: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR. In another    embodiment, the chlorotoxin is a chlorotoxin or variant thereof    comprising at least 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%,    90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to    the sequence of-   MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X is selected from K,    A and R. In another embodiment, the chlorotoxin is a chlorotoxin or    variant of thereof comprising at least 85% sequence identity to the    sequence of-   MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X is selected from K,    A and R.

In another embodiment, the chlorotoxin is BLZ-100, which is achlorotoxin variant comprising the sequence ofMCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X15 and X23 are arginineand X27 is lysine conjugated to a cyanine fluorescent label. The peptidecan be further cross-linked by four disulfide bonds formed among thecysteine residues present in the sequence.

In some aspects, the peptide is a variant of the natural peptide ofchlorotoxin but retains all eight cysteine residues of the naturalpeptide, enabling cross-linking by up to four disulfide bonds.Conservation of cysteine residues helps to preserve the secondarystructure, charge distribution, isolelectric point (pI) and otherfeatures of the natural chlorotoxin peptide because of the disulfidebonds that form between the cysteine residues.

In some aspects, the chlorotoxin peptide variant retains all eightcysteine residues of the natural peptide and has at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity with the native chlorotoxinpeptide.

In some aspects, the chlorotoxin peptide variant has eight cysteineresidues positioned so that the distances between pairs of cysteines isthe same as the distances between pairs of cysteines found in thenatural peptide, and the chlorotoxin peptide variant has at least 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%, 89%, 90%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nativechlorotoxin peptide.

In some aspects, the chlorotoxin peptide variant has eight cysteineresidues positioned so that the distances between pairs of cysteines isfunctionally equivalent or functionally similar to the distances betweenpairs of cysteines found in the natural peptide, and the chlorotoxinpeptide variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 83%, 85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with the native chlorotoxin peptide.

In some aspects, the chlorotoxin peptide variant has eight cysteineresidues positioned so that the distances between pairs of cysteinesallows for secondary structure and isolectric point of the nativechlorotoxin peptide to be preserved, and the chlorotoxin peptide varianthas at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 86%,89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitywith the native chlorotoxin peptide.

In some aspects, the chlorotoxin peptide variant has eight cysteineresidues positioned so that the distances between pairs of cysteines issufficient to allow disulfide bonds to form, and the chlorotoxin peptidevariant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%,85%, 86%, 89%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with the native chlorotoxin peptide.

In some aspects, one or more methionines of the chlorotoxin peptidevariant are replaced with other amino acids. In some aspects, one ormore methionines of the chlorotoxin peptide variant are replaced withother amino acids selected from glycine, alanine, Isoleucine, Threonine,Valine, Leucine, Serine or a combination thereof.

In some embodiments, the chlorotoxin can be a chlorotoxin variant.Chlorotoxin and chlorotoxin variants have are further described in PCTPatent Application Publication Numbers WO2006115633 and WO2011142858,which are incorporated in their entirety herein by reference.

In one embodiment, the peptide can have the following formula:H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Xaa-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Xaa-Gly-Arg-Gly-Xaa-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OHacetate salt (disulfide bonds, air oxidized), wherein Xaa is Arg, Ala,or Lys.

In another embodiment, the all peptide can have the following formula:H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Xaa-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Xaa-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OHacetate salt (disulfide bonds, air oxidized), wherein Xaa is Arg, orAla.

In another embodiment, the peptide can have the following formula:H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OHacetate salt (disulfide bonds, air oxidized).

In another embodiment, the peptide can have the following formula:H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OHacetate salt (disulfide bonds, air oxidized).

In another embodiment, the peptide can have the following formula:H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OHacetate salt (disulfide bonds, air oxidized).

In another embodiment, the peptide can have the following formula:H-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OHacetate salt (disulfide bonds, air oxidized).

In certain embodiments, the chlorotoxin and chlorotoxin variants can beconjugated to moieties, such as detectable labels (e.g., dyes) that canbe detected (e.g., visualized) in a subject. In some embodiments, thechlorotoxin and/or chlorotoxin variants can be conjugated to detectablelabels to enable tracking of the bio-distribution of a conjugatedpeptide. The detectable labels can include fluorescent dyes.Non-limiting examples of fluorescent dyes that could be used as aconjugating molecule in the present disclosure include rhodamine,rhodol, fluorescein, thiofluorescein, aminofluorescein,carboxyfluorescein, chlorofluorescein, methylfluorescein,sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol,methylrhodol, sulforhodol; aminorhodamine, carboxyrhodamine,chlororhodamine, methylrhodamine, sulforhodamine, and thiorhodamine,cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine,merocyanine, a cyanine dye (e.g., cyanine 2, cyanine 3, cyanine 3.5,cyanine 5, cyanine 5.5, cyanine 7), oxadiazole derivatives,pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyrene derivatives,cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet,oxazine 170, acridine derivatives, proflavin, acridine orange, acridineyellow, arylmethine derivatives, auramine, xanthene dyes, sulfonatedxanthenes dyes, Alexa Fluors (e.g., Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 647, Alexa Fluor 700), crystal violet, malachite green,tetrapyrrole derivatives, porphyrin, phtalocyanine, and bilirubin. Someother example dyes include near-infrared dyes, such as, but not limitedto, Cy5.5, indocyanine green (ICG), DyLight 750 or IRdye 800. In someembodiments, near infrared dyes can include cyanine dyes.

Chemotherapueutics, anti-cancer drugs, and anti-cancer agents, include,but are not limited to: radioisotopes, toxins, enzymes, sensitizingdrugs, nucleic acids, including interfering RNAs, antibodies,anti-angiogenic agents, cisplatin, anti-metabolites, mitotic inhibitors,growth factor inhibitors, paclitaxel, temozolomide, topotecan,fluorouracil, vincristine, vinblastine, procarbazine, decarbazine,altretamine, methotrexate, mercaptopurine, thioguanine, fludarabinephosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide,teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin,dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen,flutamide, leuprolide, goserelin, aminogluthimide, anastrozole,amsacrine, asparaginase, mitoxantrone, mitotane and amifostine, andtheir equivalents, as well as photo-ablation.

As used herein, the terms “about” and “approximately,” in reference to anumber, is used herein to include numbers that fall within a range of10%, 5%, or 1% in either direction (greater than or less than) thenumber unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

Suitable diagnostic agents include agents that provide for the detectionby fluorescence methods as well as methods other than fluorescenceimaging. Other suitable diagnostic agents include radiolabels (e.g.,radio isotopically labeled compounds) such as ¹²⁵ I, ¹⁴C, and ³¹P, amongothers; and magnetic resonance imaging agents.

Suitable targeting agents include antibodies, polypeptides,polysaccharides, and nucleic acids.

In another aspect of the invention, compositions that include themodified chlorotoxin peptide conjugates are provided. The compositioncan include a pharmaceutically acceptable carrier or diluent fordelivery of the modified chlorotoxin peptide conjugate. Suitablepharmaceutically acceptable carriers or diluents include saline ordextrose for injection.

In various aspects, the presently described compounds further comprise adetectable label, which can be used for the detection of thepeptide-label conjugate and the cancerous cells to which they are bound.

In various aspects, compounds of the present dislcosure have thestructure of Formula (I), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, C₁-C₆ alkoxy, C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀alkylene-(C (═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, —COOH, C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀alkylene-(C (═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁—C₆alkylene-(O—C₁—C₆ alkylene)_(n)—, —NR¹⁰—L⁴—, —NR¹⁰—C₁-C₆alkylene-NR¹¹—(C (═O)—C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are each independently selected from hydrogen, C₁-C₆ alkyl,or R¹² and R¹³ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-aryl-A⁵,-(L⁵)-heteroaryl, -(L⁵)-heteroaryl-A⁵, —NR¹⁷ R¹⁸, R¹⁴ and R¹⁹ are joinedtogether along with the other atoms to which they are attached to form a5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴ andR²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, or —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are each independently selected from hydrogen, C₁-C₆ alkyl,R¹⁴ and R¹⁹ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

one of A¹, A², A³, A⁴, or A⁵ is a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof and the others of A¹, A², A³, A⁴, or A⁵ are eachindependently absent, hydrogen, —COOH, or sulfonate.

In various aspects, the presently described compounds further comprise adetectable label, which can be used for the detection of thepeptide-label conjugate and the cancerous cells to which they are bound.

In various aspects, compounds of the present dislcosure have thestructure of Formula (XV), or a pharmaceutically acceptable saltthereof:

wherein:

R³, R⁴, R⁵, R⁶, R¹⁵, and R¹⁶ are each independently selected fromhydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate, —COOH, —SO₂—NH₂,C₁-C₆ alkoxy, alkylene-(C (═O))_(x)—, alkylene-(C (═O))_(x)—O—, orC₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, —COOH, alkylene-(C (═O))_(x)—, C₁-C₁₀alkylene-(C (═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆alkylene-(O—-C₁-C₆ alkylene)_(n)—, —NR¹⁰-L⁴-, —NR¹⁰—C₁-C₆alkylene-NR¹¹—(C (═O) —C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are each independently selected from hydrogen, C₁-C₆ alkyl,or R¹² and R¹³ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-aryl-A⁵,-(L⁵)-heteroaryl, -(L⁵)-heteroaryl-A⁵, —NR¹⁷ R¹⁸, R¹⁴ and R¹⁹ are joinedtogether along with the other atoms to which they are attached to form a5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴ andR²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, or —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are each independently selected from hydrogen, C₁-C₆ alkyl,R¹⁴ and R¹⁹ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

R²¹ and R²² are each independently selected from hydrogen, C₁-C₆ alkyl,sulfonate, or R²¹ and R²² are joined together along with the other atomsto which they are attached to form a 5-membered or 6-membered aryl;

R²³ and R²⁴ are each independently selected from hydrogen, C₁-C₆ alkyl,sulfonate, or R²³ and R²⁴ are joined together along with the other atomsto which they are attached to form a 5-membered or 6-membered aryl;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

one of A¹, A², A³, A⁴, or A⁵ is a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof and the others of A¹, A², A³, A⁴, or A⁵ are eachindependently absent, hydrogen, —COOH, or sulfonate.

In some aspects, the compounds of the present disclosure have astructure of Formula (II), or a pharmaceutically acceptable saltthereof:

In certain aspects, the present compounds have a structure of Formula(III), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R⁹ is hydrogen, sulfonate, or —COOH;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆alkylene- (O—C₁-C₆ alkylene)_(n)—, —NR¹⁰-L⁴-, —NR¹⁰—NR¹⁰—C₁-C₆alkylene-NR¹¹—(C (═O) —C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl-C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3; and

A⁴ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In other aspects, compounds of the present disclosure have a structureof Formula (IV), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R³ is selected from C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, or —COOH, or C₁-C₁₀ alkyl;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is hydrogen, sulfonate, —COOH, C₁-C₁₀ alkyl;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

A¹ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In other aspects, compounds of the present disclosure have a structureof Formula (V), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R⁵ is selected from C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

R⁹ is hydrogen, sulfonate, or —COOH, or C₁-C₁₀ alkyl;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is hydrogen, sulfonate, —COOH, or C₁-C₁₀ alkyl;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl-C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1; and

A² is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In some aspects, compounds of the present disclosure have a structure ofFormula (VI), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R⁹ is selected from C₁-C₁₀ alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is hydrogen, sulfonate, —COOH, or C₁-C₁₀ alkyl;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷R¹⁸, R¹⁴ and R¹⁹ are joined together along with the other atoms to whichthey are attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

A³ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In additional aspects, compounds of the present disclosure have astructure Formula (III), or a pharmaceutically acceptable salt thereof:

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are each independentlyselected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH, sulfonate,—COOH, —SO₂—NH₂, or C₁-C₆ alkoxy;

R⁹ is hydrogen, sulfonate, or —COOH;

L¹ is C₃-C₆ alkylene;

L² is C₁-C₁₀ alkylene;

L³ is a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆alkylene-(O—-C₁-C₆alkylene)_(n)—, —NR¹⁰-L⁴-, —NR¹⁰—C₁-C₆alkylene-NR¹¹—(C (═O)—C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene-;

L⁴ is a bond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-;

R¹⁰ is hydrogen or C₁-C₆ alkyl;

R¹¹ is hydrogen or C₁-C₆ alkyl;

R¹² and R¹³ are independently selected from hydrogen, C₁-C₆ alkyl, orR¹² and R¹³ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring;

R¹⁴ is -(L⁵)-aryl-A⁵, or -(L⁵)heteroaryl-A⁵;

L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—;

R¹⁷ and R¹⁸ are each independently hydrogen or aryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring;

n is 0, 1, 2, or 3;

m is 0, 1, 2, or 3;

p is 0, 1, 2, or 3;

q is 0, 1, 2, or 3;

x is 0 or 1;

A⁴ is hydrogen, —COOH, or sulfonate; and

A⁵ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof.

In certain aspects, A¹, A², and A³ are absent. In some aspects, A⁵ ishydrogen. In certain aspects, R³, R⁴, R⁵, and R⁶ are each independentlyC₁-C₆ alkyl. In some aspects, R³, R⁴, R⁵, R⁶ are each independentlymethyl. In certain aspects, R¹, R², R⁷, R⁸, R¹⁵, and R¹⁶ are eachindependently selected from hydrogen or sulfonate. In further aspects,R¹, R², R⁷, R⁸, R¹⁵, and R¹⁶ are each independently hydrogen. In someaspects, R¹², R¹³, R¹⁴, R¹⁹, R²⁰ are each independently hydrogen.

In certain aspects, R¹² and R¹³ join together along with the atoms towhich they are attached to form a six-membered carbocyclic ring. Inother aspects, R¹² and R¹³ join together along with the atoms to whichthey are attached to form a five-membered carbocyclic ring. In certainaspects, R¹⁴ and R¹⁹ join together along with the atoms to which theyare attached to form a six-membered carbocyclic ring. In some aspects,R¹⁴ and R²⁰ join together along with the atoms to which they areattached to form a six-membered carbocyclic ring. In certain aspects, L¹is C₃-C₆ alkylene. In other aspects, L¹ is C3-Cs alkylene. In stillother aspects, L¹ is propylene. In still other aspects, L¹ is butylene.In other aspects, L¹ is pentylene. In some aspects, L² is C₃-C₆alkylene. In other aspects, L² is propylene. In still other aspects, L²is butylene. In other aspects, L² is pentylene. In some aspects, R⁹ issulfonate. In other aspects, R⁹ is hydrogen. In some aspects, R¹⁴ ishydrogen. In other aspects, R¹⁴ is -(L⁵)-aryl. In still other aspects,R¹⁴ is -(L⁵)—aryl-A⁵.

In some aspects, R¹ is hydrogen. In certain aspects, R² is hydrogen. Insome aspects, R³ is methyl. In certain aspects, R⁴ is methyl. In someaspects, R⁵ is methyl. In certain aspects R⁶ is methyl. In some aspects,R⁷ is hydrogen. In certain aspects, R⁸ is hydrogen. In some aspects, R¹²is hydrogen. In certain aspects, R¹³ is hydrogen. In some aspects, R¹⁴is hydrogen. In certain aspects, R¹⁹ is hydrogen. In some aspects, R²⁰is hydrogen. In certain aspects, R¹⁰ is hydrogen. In some aspects, R¹¹is hydrogen.

In some aspects, R¹⁷ and R¹⁸ are independently phenyl. In some aspects,L³ is selected from a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-,—O—NR¹⁰—, or —NR¹⁰—L⁴—. In further aspects, L³ is a bond.

In some aspects, L⁴ is -heterocyclyl- or -heterocyclyl- C₁-C₆ alkylene-.In further aspects, L⁴ is -piperizinyl-(C₁-C₆ alkylene)-. In stillfurther aspects, L⁴ is

In some aspects, p is 1. In certain aspects, q is 1.

In some aspects, the compound has the structure of any one of Formulas(VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV):

In some aspects, one of A¹, A², A³, A⁴, or A⁵ is a polypeptide having atleast 87% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR ora fragment thereof. In further aspects, one of A¹, A², A³, A⁴, or A⁵ isa polypeptide having at least 90% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. In stillfurther aspects, one of A¹, A², A³, A⁴, or A⁵ is a polypeptide having atleast 92% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR ora fragment thereof. In still further aspects, one of A¹, A², A³, A⁴, orA⁵ is a polypeptide having at least 95% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. In stillfurther aspects, one of A¹, A², A³, A⁴, or A⁵ is a polypeptide having atleast 97% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR ora fragment thereof. In still further aspects, one of A¹, A², A³, A⁴, orA⁵ is a polypeptide having 100% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. In stillfurther aspects, one of A¹, A², A³, A⁴, or A⁵ is a polypeptide havingthe sequence MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof

In some aspects, the fragment of A¹, A², A³, A⁴, or A⁵ has a length ofat least 25 amino acid residues. In further aspects, the fragment of A¹,A², A³, A⁴, or A⁵ has a length of at least 27 amino acid residues. Instill further aspects, the fragment of A¹, A², A³, A⁴, or A⁵ has alength of at least 29 amino acid residues. In still further aspects, thefragment of A¹, A², A³, A⁴, or A⁵ has a length of at least 31 amino acidresidues. In still further aspects, the fragment of A¹, A², A³, A⁴, orA⁵ has a length of at least 33 amino acid residues.

In some aspects, one of A¹, A², A³, A⁴, or A⁵ is a a polypeptide havingat least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCRor a fragment thereof having the tumor cell binding affinity of nativechlorotoxin. In certain aspects, one of A¹, A², A³, A⁴, or A⁵ is a apolypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof havingessentially the same the tumor cell binding affinity of nativechlorotoxin. In some aspects, one of A¹, A², A³, A⁴, A⁵ is a apolypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof having thetumor cell binding affinity of native chlorotoxinwherein one of A¹, A²,A³, A⁴, or A⁵ has a sequence selected from SEQ ID NOS: 1-481.

In some aspects, the polypeptide comprises at least one lysine aminoacid residue. In certain aspects, the polypeptide comprises a singlelysine amino acid residue. In some aspects, the polypeptide comprisesone, two, or three lysine amino acid residues. In some aspects, thepolypeptide comprises a lysine residue at the position corresponding toK-27 of native chlorotoxin. In some aspects, the polypeptide comprises alysine residue at the position corresponding to K-23 of nativechlorotoxin. In some aspects, the polypeptide comprises a lysine residueat the position corresponding to K-15 of native chlorotoxin.

In some aspects, one or more of the amino acids of the polypeptide issubstituted with a non-naturally occurring amino acid residue. Infurther aspects the non-naturally occurring amino acid residue is acitrulline amino acid residue. In still further aspects, L³ is attachedto A⁴ at a citrulline amino acid residue of the polypeptide.

In some aspects, L³ is attached to A⁴ at a lysine amino acid residue ofthe polypeptide. In certain aspects, L³ is attached to A⁴ at theN-terminus of the polypeptide. In some aspects, L³ is attached to A⁴ atthe C-terminus of the polypeptide. In some aspects, the R³ is attachedto A¹ at a lysine amino acid residue of the peptide, a citrulline aminoacid residue of the polypeptide, the N-terminus of the polypeptide, orthe C-terminus of the polypeptide. In some aspects, the R⁵ is attachedto A² at a lysine amino acid residue of the polypeptide, a citrullineamino acid residue of the polypeptide, the N-terminus of thepolypeptide, or the C-terminus of the polypeptide. In some aspects, theR⁹ is attached to A³ at a lysine amino acid residue of the polypeptide,a citrulline amino acid residue of the polypeptide, the N-terminus ofthe polypeptide, or the C-terminus of the polypeptide. In some aspects,the aryl is attached to A⁵ at a lysine amino acid residue of thepolypeptide, a citrulline amino acid residue of the polypeptide, theN-terminus of the polypeptide, or the C-terminus of the polypeptide.

In some aspects, the compound has the structure of any one of compounds1 to 721 as found in Tables 2-13.

In some aspects, the compound is conjugated to polyethylene glycol(PEG), hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer,a zwitterionic water soluble polymer, a water soluble poly(amino acid),an albumin derivative, or a fatty acid.

In some aspects, the polypeptide has an isoelectric point of from 7.5 to9.0. In some aspects, the polypeptide has an isoelectric point of from8.0 to 9.0. In some aspects, the polypeptide has an isoelectric point offrom 8.5 to 9.0. In some aspects, the polypeptide is basic and has anisoelectric point of greater than 7.5.

In some aspects, the polypeptide comprises at least eight cysteine aminoacid residues. In some aspects, the polypeptide comprises eight cysteineamino acid residues. In some aspects, the polypeptide comprises fourdisulfide bonds. In some aspects, the polypeptide comprises from six toseven cysteine amino acid residues. In some aspects, the polypeptidecomprises three disulfide bonds. In some aspects, the spacing betweenthe cysteine amino acid residues in the polypeptide is essentially thesame as in native chlorotoxin. In some aspects, the distribution ofcharge on the surface of the polypeptide is essentially the same as innative chlorotoxin.

In some aspects, one or more of the methionine amino acid residues isreplaced with an amino acid residue selected from isoleucine, threonine,valine, leucine, serine, glycine, alanine, or a combination thereof.

In some aspects, the compound is capable of passing across the bloodbrain barrier. In some aspects, the compound further comprises atherapeutic agent attached to A. In further aspects, the therapeuticagent is a cytotoxic agent.

In various aspects, the present disclosure provides a compositioncomprising a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof, wherein when the composition is intravenouslyadministering to a human subject at a dose of from 1 mg to 30 mg, thecomposition produces in the human subject an average maximum compoundblood plasma concentration (average C_(max))of at least from 110 ng/mLto240 ng/mL per each 1 mg dosage of the compound administered.

In some aspects, the compound of the composition is any suitablecompound described in the present disclosure.

Certain exemplary compounds falling within the scope of these genusesare provided below in Tables 2 to 13, including both the peptide portion(indicated by A) and the detectable label portion.

TABLE 2 Exemplary compounds according to the present disclosure. No.Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

A = MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR (SEQ ID NO: 9) (attached atK-27)

TABLE 3 Exemplary compounds according to the present disclosure. No.Structure 61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

A = MCMPCFTTDHQMARACDDCCGGAGRG K CYGPQCLCR (SEQ ID NO: 5) (attached atK-27)

TABLE 4 Exemplary compounds according to the present disclosure. No.Structure 121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

A = MCMPCFTTDHQMARRCDDCCGGAGRG K CYGPQCLCR (SEQ ID NO: 6) (attached atK-27)

TABLE 5 Exemplary compounds according to the present disclosure. No.Structure 181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

A = MCMPCFTTDHQMARACDDCCGGRGRG K CYGPQCLCR (SEQ ID NO: 8) (attached atK-27)

TABLE 6 Exemplary compounds according to the present disclosure. No.Structure 241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

A = MCMPCFTTDHQMARACDDCCGG K GRGACYGPQCLCR (SEQ ID NO: 11) (attached atK-23)

TABLE 7 Exemplary compounds according to the present disclosure. No.Structure 301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

A = MCMPCFTTDHQMARRCDDCCGG K GRGACYGPQCLCR (SEQ ID NO: 12) (attached atK-23)

TABLE 8 Exemplary compounds according to the present disclosure. No.Structure 361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

711

712

713

714

715

716

717

718

719

720

A = MCMPCFTTDHQMAR K CDDCCGGAGRGACYGPQCLCR (SEQ ID NO: 13) (attached atK-15)

TABLE 9 Exemplary compounds according to the present disclosure. No.Structure 411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

A = MCMPCFTTDHQMAR K CDDCCGGRGRGACYGPQCLCR (SEQ ID NO: 16) (attached atK-15)

TABLE 10 Exemplary compounds according to the present disclosure. No.Structure 471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

A = MCMPCFTTDHQMARACDDCCGG K GRGRCYGPQCLCR (SEQ ID NO: 20) (attached atK-23)

TABLE 11 Exemplary compounds according to the present disclosure. No.Structure 531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

A = MCMPCFTTDHQMARRCDDCCGG K GRGRCYGPQC (SEQ ID NO: 21) (attached atK-23)

TABLE 12 Exemplary compounds according to the present disclosure. No.Structure 591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

A = MCMPCFTTDHQMAR K CDDCCGGAGRGRCYGPQCLC (SEQ ID NO: 22) (attached atK-15)

TABLE 13 Exemplary compounds according to the present disclosure. No.Structure 651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704

705

706

707

708

709

710

A = MCMPCFTTDHQMAR K CDDCCGGRGRGRCYGPQCLCR (SEQ ID NO: 25) (attached atK-15)

In certain aspects, the presently described peptides are conjugated tomoieties, such as detectable labels (e.g., dyes or radiolabels) that aredetected (e.g., visualized) in a subject. In some aspects, thechlorotoxin and/or chlorotoxin variants is conjugated to detectablelabels to enable tracking of the bio-distribution of a conjugatedpeptide. The fluorescent moiety is covalently coupled to the chlorotoxinto allow for the visualization of the conjugate by fluorescence imaging,either directly or through a linker as described herein and known to oneof ordinary skill in the art.

In some aspects, the fluorescent label has emission characteristics thatare desired for a particular application. For example, the fluorescentlabel is a fluorescent dye that has a emission wavelength maximumbetween a range of 500 nm to 1100 nm, between a range of 600 nm to 1000nm, between a range of 600 to 800 nm, between a range of 650 nm to 850nm, or between a range of 700 nm to 800 nm. For another example, thefluorescent label is a fluorescent dye that has a emission wavelengthmaximum between a range of about 500 nm to about 1100 nm, between arange of about 600 nm to about 1000 nm, between a range of about 600 toabout 800 nm, between a range of about 650 nm to about 850 nm, orbetween a range of about 700 nm to about 800 nm. One of ordinary skillin the art will appreciate the various dyes that are used as detectablelabels and that have the emission characteristics above.

Some other examplary dyes include near-infrared dyes, such as, but notlimited to, DyLight-680, DyLight-750, VivoTag-750, DyLight-800,IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG). In someaspects, near infrared dyes often include cyanine dyes. Additionalnon-limiting examples of fluorescent dyes for use as a conjugatingmolecule in the present disclosure include acradine orange or yellow,Alexa Fluors and any derivative thereof, 7-actinomycin D,8-anilinonaphthalene-1-sulfonic acid, ATTO dye and any derivativethereof, auramine-rhodamine stain and any derivative thereof,bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene,5,12-bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein,carbodyfluorescein and any derivative thereof,1-chloro-9,10-bis(phenylethynyl)anthracene and any derivative thereof,DAPI, DiOC6, DyLight Fluors and any derivative thereof, epicocconone,ethidium bromide, FlAsH-EDT₂, Fluo dye and any derivative thereof,FluoProbe and any derivative thereof, Fluorescein and any derivativethereof, Fura and any derivative thereof, GelGreen and any derivativethereof, GelRed and any derivative thereof, fluorescent proteins and anyderivative thereof, m isoform proteins and any derivative thereof suchas for example mCherry, hetamethine dye and any derivative thereof,hoeschst stain, iminocoumarin, indian yellow, indo-1 and any derivativethereof, laurdan, lucifer yellow and any derivative thereof, luciferinand any derivative thereof, luciferase and any derivative thereof,mercocyanine and any derivative thereof, nile dyes and any derivativethereof, perylene, phloxine, phyco dye and any derivative thereof,propium iodide, pyranine, rhodamine and any derivative thereof,ribogreen, RoGFP, rubrene, stilbene and any derivative thereof,sulforhodamine and any derivative thereof, SYBR and any derivativethereof, synapto-pHluorin, tetraphenyl butadiene, tetrasodium tris,Texas Red, Titan Yellow, TSQ, umbelliferone, violanthrone, yellowfluroescent protein and YOYO-1. Other Suitable fluorescent dyes include,but are not limited to, fluorescein and fluorescein dyes (e.g.,fluorescein isothiocyanine or FITC, naphthofluorescein,4′,5′-dichloro-2,7′-dimethoxyfluorescein, 6-carboxyfluorescein or FAM,etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes,phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g.,carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.),Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes(e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e.g., ALEXAFLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXAFLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXAFLUOR 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R⁶G, BODIPY TMR,BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, etc.), IRDyes(e.g., IRD40, IRD 700, IRD 800, etc.), and the like. In some aspects,conjugates of the present disclosure comprise other dyes, including butnot limited to those provided below in Table 14.

TABLE 14 Exemplary fluorescent reporter molecules with peak absorbance(Abs.) and emission (Em.) wavelengths specifiec (in nanometers). PeakPeak Dye Abs. Em. Methoxycoumarin 360 410 Fluospheres Blue 356 412Cascade Blue 377 420 PBFI 360 420 DyeLight 405 400 420 Cascade Blue 400420 Alexa Fluor 405 401 421 Alexa Fluor 405 401 421 LysoTracker Blue 373422 LysoSensor Blue 374 424 AMCA 345 425 True Blue 365 4257-amino-4-methylcoumarin 351 430 (AMC) Phorwite AR 360 430 DyLight 350353 432 Uvitex SFC 365 435 4-methylumbelliferone 360 440 CellTraceCalcein Blue 373 440 Calcofluor White 350 440 Fast Blue 360 440LysoSensor Yellow/Blue 329 440 (pH 8.0) LysoSensor Yellow/Blue 329 440(pH 8.0) LysoSensor Yellow/Blue 329 440 (pH 8.0) LysoSensor Yellow/Blue329 440 (pH 8.0) Alexa Fluor 350 346 442 AMCA-X 353 442 LIVE/DEADFixable Blue 344 442 Dead Cell Stain Thiolyte 378 483 SYTO 45 452 484SYTO 45 452 484 SYTO 45 452 484 SYTO 45 452 484 SYTO 45 452 484 Hoechst33258 345 487 AmCyan 548 489 Auramine O 445 500 SYTO 9 482 500 SYTO 9482 500 SYTO 9 482 500 SYTO 9 482 500 SYTO 9 482 500 DiO 484 501 DiO 484501 DiO 484 501 LysoSensor Green 448 503 LysoSensor Green 448 503LysoSensor Green 448 503 LysoSensor Green 448 503 LysoSensor Green 448503 SYTO 13 487 505 LysoSensor Green (pH 5) 442 505 SYTO 13 487 505 SYTO13 487 505 SYTO 13 487 505 SYTO 13 487 505 DiO (Vybrant DiO) 489 506 HCSLipidTox Green 498 506 LIVE/DEAD Fixable Green 498 506 LIVE/DEAD FixableGreen 498 506 ATTO 465 453 507 CellLights GFP 488 507 Evans Blue 460 515rsGFP (red shifted GFP, 498 516 S65T) CellTracker Violet BMQC 415 516HCS CellMask Green 493 516 CellTracker Violet BMQC 415 516 CellTrackerViolet BMQC 415 516 CellTracker Violet BMQC 415 516 CellTracker VioletBMQC 415 516 HCS CellMask Green 493 516 5-carboxyfluorescein 492 518(5-FAM) ActinGreen (Alexa Fluor 488 phalloidin) 496 518 Alexa Fluor 488496 518 Click-iT EdU Alexa Fluor 488 496 518 DyLight + C110 488 493 518Fluoro-Emerald 494 518 Aiexa Fluor 488 496 518 Carboxyfluorescein(5-FAM) 492 518 Aiexa Fluor 488 496 518 Carboxyfluorescein (5-FAM) 492518 CellRox Green 485 520 FITC (Fluorescein) 492 520 Fluor-X 494 520Rhodamine 110 496 520 SYTO 16 490 520 FITC 492 520 Rhodamine 110 496SYTO 16 490 520 FITC 492 520 Rhodamine 110 496 520 SYTO 16 490 520 Qdot525 UV 525 SYTO 11 506 525 Qdot 525 UV 525 Qdot 525 UV 525 AcridineOrange + DNA 500 526 LIVE/DEAD Fixable Green 498 526 Surf Green EX 469526 Acridine Orange + DNA 500 526 Acridine Orange + DNA 500 526 AcridineOrange + DNA 500 526 Acridine Orange (+DNA) 500 526 ThiolTracker Violet405 526 ThiolTracker Violet 405 526 ThiolTracker Violet 405 526ThiolTracker Violet 405 526 Acridine Orange (+DNA) 500 526 ThiolTrackerViolet 405 526 SYTO RNASelect 503 527 EYFP 514 527 SYTO RNASelect 503527 SYTO RNASelect 503 527 SYTO RNASelect 503 527 SYTO RNASelect 503 527Rhodamine 123 507 529 YFP 512 529 F2N12S 405 530, 585 F2N12S 405 530,585 F2N12S 405 530, 585 F2N12S 405 530, 585 Calcein 494 540 CalciumGreen-1 506 540 Catskill Green 540 482 540 CellTracker Green 490 540CFDA 494 540 CFP 434 540 Cy2 492 540 CyQUANT Direct 500 540 (CyQUANT GR)DAF-FM 493 540 Emerald Green 490 540 Fluo-3 506 540 Fluo-4 494 540H2DCFDA (H2- 504 540 DCF,DCFR) Alexa Fluor 430 434 540 Alexa Fluor 430432 540 BCECF (pH 5.2) 499 540 Calcein 494 540 CellTracker Green CMFDA490 540 CFP 434 540 Cy2 492 540 CyQUANT Direct 500 540 DAF-FM 493 540Fluo-4 494 540 Alexa Fluor 430 432 540 BCECF (pH 5.2) 499 540 Calcein494 540 CellTracker Green CMFDA 490 540 CFP 434 540 Cy2 492 540 CyQUANTDirect 500 540 Alexa Fluor 430 432 540 BCECF (pH 5.2) 499 540 PacificOrange 440 551 Pacific Orange 440 551 Pacific Orange 440 551 PacificOrange 440 551 Pacific Orange 440 551 Pacific Orange 440 551 mBanana 540553 ER-Tracker Blue-White 371 554 DPX Alexa Fluor 532 532 554 FocalCheckDouble Orange 540 555 HEX 533 558 Fluospheres Orange 539 560 mHoneydew478 561 Vybrant DyeCycle Orange 518 562 ActinRed 555 (rhodamin 540 565pphalloidin) Alexa Fluor 555 555 565 CellRox Orange 545 565 Qdot 565 UV565 Qdot 565 UV 565 DiI (CellTracker DiI) 551 568 mOrange 548 568 OFP546 568 Bodipy TMR 544 569 Cy3 552 570 PO-PRO-3 539 570 SYTOX Orange 567570 CellMask Orange 556 571 Alexa Fluor 546 561 572 POPO-3 532 573TurboRFP 553 574 Calcium Orange 549 575 CellTracker Orange 547 575 Qdot585 UV 585 DsRed Monomer 556 586 pHrodo Red 559 586 Carboxy SNARF-1 548587 pHrodo Red 559 587 SpectrumOrange 559 588 DsRed2 563 588 DiA 456 590DiA 456 590 DiA 456 590 DiA 456 590 DiA 456 590 DiA 456 590 DiA 456 590DiA 456 590 rhodamine Red-X 572 591 CellTrace calcein red-orange 575 592LysoTracker Red 573 592 Sulforhodamine 101 578 593 sulforhodamine 101577 593 ROX (6-ROX) 568 595 2-dodecylresorufin 582 595 Cy3.5 579 597 Cy3.5 581 597 MitoTracker Red CMXRos 578 597 BOBO-3 570 602 EthidiumBromide 521 602 X-rhod-1 579 602 BOBO-1 570 602 BOBO-1 570 602 BOBO-1570 602 5-ROX 577 603 Alexa Fluor 568 578 603 Qdot 605 UV 605 Qdot 625UV 625 Qdot 625 UV 625 FM 1-43 510 626 FM 1-43 510 626 FM 1-43 510 626FM 1-43 510 626 FM 1-43 510 626 FM 1-43 510 626 FM 1-43 510 626 FM 1-43510 626 YO-PRO-3 612 628 Alexa Fluor 610 610 629 Magic Red 570 630 CTCFormazan 450 630 CTC Formazan 450 630 YOYO-3 612 631 Katushka (TurboFP635) 588 635 mKate 588 635 SYTO 17 620 635 Di-8 ANEPPS 468 635 Di-8ANEPPS 468 635 Di-8-ANEPPS 465 635 Di-8-ANEPPS 465 635 Di-8-ANEPPS 465635 Di-8-ANEPPS 465 635 Di-8-ANEPPS 465 635 Di-8-ANEPPS 465 635Di-8-ANEPPS 465 635 Nile Red 551 636 Nile red (triglyceride) 552 636Nile red (triglyceride) 552 636 Nile red (triglyceride) 552 636 Fura Red(high Ca2+) 436 637 Nile Red phospholipid 551 638 DDAO 648 658 DyLight633 638 658 SYTOX Red 640 658 ATTO 635 635 658 APC (Allophycocyanin) 651660 MitoTracker Deep Red FM 641 661 NucRed Dead 647 642 661 TOTO-3 642661 BODIPY 650/665 647 665 CellRox Deep Red 640 665 LIVE/DEAD FixableFar 650 665 Red Cy5 648 666 Lysotracker Deep Red 647 668 Alexa Fluor 647650 670 Click-iT Alexa Fluor 647 650 670 DiD (Vybrant DiD) 645 670 HCSCellMask Deep Red stain 649 670 ATTO 647 644 670 Fura Red (−Ca2+) 473670 Fura Red (−Ca2+) 473 670 Fura Red (−Ca2+) 473 670 Fura Red (−Ca2+)473 670 Fura Red (−Ca2+) 473 670 DyLight 649 654 673Carboxynaphthofluorescein 600 674 PerCP 488 675 CellMask Deep Red plasma658 676 membrane stain DRAQ5 650 680 SYTO 60 649 681 SYTO 62 650 681SYTO 60 650 681 FM 4-64 558 734 Cy7 745 766 LIVE/DEAD Fixable near- 750775 IR CellVue NIR780 743 776 DyLight 750 752 778 IRDye 800CW 774 789XenoLight CF770 770 797 Qdot 800 UV 800 Qdot 800 UV 800 IndocyanineGreen 768 807 Y66H 360 442 ABQ 344 445 BFP 382 448 BFP 382 4487-hydroxy-4- 360 449 methylcoumarin SpectrumBlue 405 449 DiFMU (pH 9.0)357 450 sgBFP (Super Glow BFP) 387 450 SpectrumBlue 400 450 CellTraceCalcein Violet 401 451 DAPI 345 455 NucBlue Fixed Cell Stain 345 455Pacific Blue 405 455 Pacific Blue 410 455 PO-PRO-1 435 455 PO-PRO-1 435455 POPO-1 434 456 POPO-1 434 456 TagBFP 402 457 Marina Blue 365 460SITS 365 460 Thioflavin TCN 350 460 Monochlorobimane (mBCI) 380 461Quinine Sulfate 349 461 Acridine 362 462 CellLights CFP 434 477 ECFP 434477 CFP 434 477 1,8-ANS 372 480 SYTOX Blue 444 480 SYTOX Blue 444 480Hoechst 33342 347 483 NucBlue Live Cell Stain 347 483 CellEventCaspase-3/7 Green 488 507 Diversa Green-FP 484 507 GFP (EGFP) 488 507S65C 479 507 YO-PRO-1 491 507 GFP 488 507 YO-PRO-1 491 507 GFP 488 507YO-PRO-1 491 507 GFP 488 507 YO-PRO-1 491 507 Premo FUCCI Cell Cycle 474509 Sensor (S/G2/M phases) sgGFP (Super Glow GFP) 474 509 wtGFP (wildtype GFP, 475 509 non-UV excitation) YOYO-1 491 509 YOYO-1 491 509YOYO-1 491 509 YOYO-1 491 509 YOYO-1 491 509 HPTS (Solvent Green 7) 455510 Nitrobenzoxadiazole 465 510 565L 484 510 LysoTracker Green 504 511565T 488 511 LysoTracker Green 504 511 LysoTracker Green 504 511MitoTracker Green FM 490 512 MitoTracker Green FM 490 512 MitoTrackerGreen FM 490 512 MitoTracker Green FM 490 512 FluoSpheres Yellow-Green501 513 Evans Blue 460 515 SYTO 16 490 520 FITC 492 520 Rhodamine 110496 520 SYTO 16 490 520 SYBR Green I 497 521 SYBR Green I 497 521 SYBRGreen I 497 521 SYBR Green I 497 521 SYBR Green I 497 521 Quant-iTPicoGreen 502 522 Spectru mgreen 498 522 NucGreen Dead Cell Stain 504523 Rhodamine Green 497 523 Rhodol Green 496 523 SYTOX Green 504 523Rhodamine Green 497 523 Rhodamine Green 497 523 Rhodamine Green 497 523Neurotrace 500/525 Green 497 524 Oregon Green 488 498 524 SYBR Safe 507524 NeuroTrace 500/525 Nissl stain 497 524 Oregon Green 488 498 524NeuroTrace 500/525 Nissl stain 497 524 Oregon Green 488 498 524NeuroTrace 500/525 Nissl stain 497 524 NeuroTrace 500/525 497 524 Nisslstain Oregon Green 488 498 524 Dansyl 335 525 Fluoro-Jade B 480 525F2N12S 405 530, 585 F2N12S 405 530, 585 F2N12S 405 530, 585 MagnesiumGreen 506 530 NBD Amine 450 530 TO-PRO-1 515 530 TOTO-1 513 531 OregonGreen 514 512 532 Sodium Green 506 532 Vybrant DyeCycle Green 505 532pHrodo Green 509 533 NBD-X 467 538 NBD-X 467 538 NBD-X 467 538 NBD-X 467538 NBD-X 467 538 NBD-X 467 538 NBD-X 467 538 SYBR Gold 495 539 SYBRGold 495 539 SYBR Gold 495 539 SYBR Gold 495 539 SYBR Gold 495 539 AlexaFluor 430 432 540 Auramine 460 540 Aurophosphine 470 540 BCECF 499 540BODIPY 492/515 490 540 BODIPY 505/515 502 540 BODIPY FL 502 540 BTC 464540 CFP 434 540 Cy2 492 540 Alexa Fluor 430 432 540 BCECF (pH 5.2) 499540 Alexa Fluor 430 432 540 BCECF (pH 5.2) 499 540 Alexa Fluor 430 432540 BCECF (pH 5.2) 499 540 Calcein 494 540 CellTracker Green CMFDA 490540 CFP 434 540 Cy2 492 540 CyQUANT Direct 500 540 DAF-FM 493 540 Fluo-4494 540 TET 520 541 TET 521 542 Lucifer Yellow 423 543 Qdot 545 UV 543Lucifer Yellow 423 543 Lucifer Yellow 423 543 Lucifer Yellow 423 543Lucifer Yellow 423 543 Lucifer Yellow 423 543 Lucifer Yellow 423 543Lucifer Yellow 423 543 Lucifer yellow 428 544 Lucifer Yellow 428 544Lucifer yellow 428 544 Eosin 524 545 JOJO-1 529 545 Qdot 545 UV 545 Qdot545 UV 545 Auramine O 460 550 LIVE/DEAD Fixable Yellow 405 575 LIVE/DEADFixable Yellow 405 575 LIVE/DEAD Fixable Yellow 405 575 LIVE/DEADFixable Yellow 405 575 LIVE/DEAD Fixable Yellow 405 575 LIVE/DEADFixable Yellow 405 575 DyLight 594 562 576 MitoTracker Orange 551 576CMTMRos (MitoTracker Orange CM-H2TMRos) Phycoerythrin 567 576 (PE,R-phycoerythrin) Rhod-2 551 576 Rhodamine Phalloidin 557 576 X-Rhod-1570 576 DsRed-Express 557 579 Rhodamine Red 560 580 TAMRA 565 580Tetramethylrhodamine (TRI 555 580 TC) dTomato 554 581 DsRed2 563 582Amplex Ultra Red 567 582 Amplex Red 571 583 Amplex UltraRed 568 583Amplex Red 570 583 Premo FUCCI Cell Cycle 555 584 Sensor (G1 phase)TagRFP 555 584 CellLights RFP 552 585 mTangerine 568 585 Resorufin 570585 RFP 552 585 Qdot 585 UV 585 Qdot 605 UV 605 BOBO-3 571 606 CalciumCrimson 589 608 Fluospheres Red 577 608 microspheres ReAsH (TC-ReAsH)593 608 CellTracker Red 585 612 LIVE/DEAD Fixable Red 593 613CellTracker Red CMTPX 584 613 LIVE/DEAD 595 613 Fixable Red Dead Cellstain DiA (FAST DiA) 491 613 DiA 491 613 HCS CellMask Red stain 587 614HCS LipidTox Red 582 615 HCS LipidTOX Red 582 615 mCherry 587 615 TexasRed 592 615 Ethidium Homodimer-1 530 618 (EthD-1) Propidium Iodide (PI)530 618 Alexa Fluor 594 590 618 Click-iT Alexa Fluor 594 590 618 DyLight594 593 618 SYPRO Ruby 450 618 SYPRO Ruby 450 618 SYPRO Ruby 450 618SYPRO Ruby 450 618 SYPRO Ruby 450 618 SYPRO Ruby 450 618 Bodipy TR-X 588621 CellTrace BODIPY TR 597 625 methyl esther mRaspberry 598 625 SYTO 17619 638 Bodipy 630/650-X 625 641 BODIPY 630/650X 626 641 7-AAD 549 644HCS NuclearMask Red 624 644 HCS NuclearMask Red 622 644 SYTO 59 621 644SYTO 59 622 645 Fluospheres Crimson 620 646 microspheres FluoSpherescrimson 621 646 microspheres SYTOX AADvanced dead cell stain 546 647Alexa Fluor 635 634 647 HcRed 594 649 mPlum 590 649 SYTO 61 619 649Alexa Fluor 633 631 650 Acridine Orange + RNA 460 650 Acridine Orange +RNA 460 650 Acridine Orange(+RNA) 460 650 Acridine Orange(+RNA) 460 650HCS LipidTOX Deep Red 634 652 Fura Red ( +Ca2+) 436 655 Fura Red (+Ca2+) 436 655 Fura Red ( +Ca2+) 436 655 Fura Red ( +Ca2+) 436 655 Qdot655 UV 655 Fura Red (+Ca2+) 436 655 Fura Red (+Ca2+) 436 655 Qdot 655 UV655 FxCycle Far Red 641 657 TO-PRO-3 642 657 FluoSpheres dark red 657683 microspheres ATTO 655 663 683 FluoSpheres Dark Red 656 683fluorescent microspheres NucRed Live 647 638 686 Vybrant DyeCycle Ruby638 686 HCS NuclearMask Deep Red 635 687 Cy5.5 672 690 Alexa Fluor 660663 691 Alexa Fluor 660 663 691 Cy5.5 678 696 DY-675 675 699 IRDye 700Phosphoramidite 691 699 ATTO 680 680 700 Alexa Fluor 680 679 702 HiLyteFluor 680 688 702 Qdot 705 Nanocrystals 300 702 Alexa Fluor 680 679 704DyLight 680 676 705 Qdot 705 UV 705 Qdot 705 UV 705 Quasa 705 688 706IRDye 680 NHS Ester 683 710 RH 795 530 712 RH 795 530 712 RH 795 530 712RH 795 530 712 RH 795 530 712 Alexa Fluor 700 696 719 ATTO 700 699 719FM 4-64 558 734 FM 4-64 558 734 FM 4-64 558 734

In some other aspects, the conjugate compounds include achemiluminescent compound, colloidal metal, luminescent compound,enzyme, radioisotope, or paramagnetic labels.

In certain aspects, the conjugates of the present disclosure areconjugated to radioactive isotopes instead of or in addition to othertypes of detectable agents. Certain isotopes sutable for use in thepresent compounds include, but not limited to, iodine-131, iodine-125,bismuth-212, bismuth-213, lutetium-177, rhenium-186, rhenium-188,yttrium-90, astatine-211, phosphorus-32 and/or samarium-153. In someaspects, the conjugates of the present disclosure contain one or moreatoms having an atomic mass or mass number different from the atomicmass or mass number usually found in nature, including but not limitedto hydrogen, carbon, fluorine, phosphorous, copper, gallium, yttrium,technetium, indium, iodine, rhenium, thallium, bismuth, astatine,samarium, and lutetium (for example, ³H, ³H, ¹³C, ¹⁴C, ¹⁸F, ³²P, ³⁵S,⁶⁴Cu, ⁶⁷Ga, ⁹⁰Y, ^(99M)Tc, ¹¹¹In, ¹²⁵I, ¹²³I, ¹³⁵, ¹⁸⁶Re, ¹⁸⁷Re, ²⁰¹TI,²¹²Bi, ²¹¹At, ¹⁵³Sm and/or ¹⁷⁷Lu). In other aspects, the conjugates ofthe present disclosure are labeled with a paramagnetic metal ion that isa good contrast enhancer in Magnetic Resonance Imaging (MRI). Examplesof such paramagnetic metal ions include, but are not limited to,gadolinium III (Gd3+), chromium 111 (Cr3+), dysprosium III (Dy3+), iron111 (Fe3+), manganese II (Mn2+), and ytterbium III (Yb3+). In certainembodiments, the labeling moieties comprises gadolinium III (Gd3+).

In some aspects, the conjugates of the present disclosure are conjugatedto biotin. In addition of extension of half-life, biotin also acts as anaffinity handle for retrieval of the peptides from tissues or otherlocations. In one aspect, the conjugates are conjugated, e.g., to abiotinidase resistant biotin with a PEG linker (e.g.,NHS-dPEG4-Biotinidase resistant biotin). In some aspects, fluorescentbiotin conjugates that can act both as a detectable label and anaffinity handle are used. Non-limiting examples of commerciallyavailable fluorescent biotin conjugates include Atto 425-Biotin, Atto488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto 565-Biotin, Atto590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto 740-Biotin,fluorescein biotin, biotin-4-fluorescein, biotin-(5-fluorescein)conjugate, and biotin-B-phycoerythrin, alexa fluor 488 biocytin, alexaflour 546, alexa fluor 549, lucifer yellow cadaverine biotin-X, Luciferyellow biocytin, Oregon green 488 biocytin, biotin-rhodamine andtetramethylrhodamine biocytin.

Linkers. In some aspects, the peptides of the present disclosure aredirectly conjugated to a detectable label, such as a dye, fluorescentmoiety or the like such that no additional amino acids, carbohydrates,nucleic acids, polymers, organic chains, or the like are added to thechlorotoxin or chlorotoxin variant and/or the dye, fluorescent moiety orthe like to comprise the chlorotoxin conjugates described herein. Insome other aspects, a linker is used to conjugate the chlorotoxin orchlorotoxin variant is not directly conjugated to a dye, fluorescentmoiety or the like such that additional amino acids, carbohydrates,nucleic acids or the like are added to the chlorotoxin or chlorotoxinvariant and/or the dye, fluorescent moiety or the like to comprise thechlorotoxin conjugates described herein. A “linker” as used hereinrefers to at least one compound comprising two functional groups thatare capable of reacting specifically with other moieties to formcovalent or non-covalent linkages. Such moieties include, but are notlimited to, the side groups on natural or non-natural amino acids orpeptides which contain such natural or non-natural amino acids. By wayof example, a linker has a functional group reactive with a group on afirst peptide, and another functional group which is reactive with agroup on a second peptide, whereby forming a conjugate that includes thefirst peptide, the linker and the second peptide. Many procedures andlinker molecules for attachment of various compounds to peptides areknown. See, e.g., European patent application Ser. No. 188,256; U.S.Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and4,569,789 which are incorporated by reference herein in their entirety.

The term “linkage,” as used herein refers to a bond or a chemical moietyformed from a chemical reaction between the functional group of a linkerand another molecule. Such bonds include, but are not limited to,covalent linkages and non-covalent bonds, while such chemical moietiesinclude, but are not limited to, esters, carbonates, imines phosphateesters, hydrazones, acetals, orthoesters, peptide linkages, andoligonucleotide linkages. Hydrolytically stable linkages means that thelinkages are substantially stable in water and do not react with waterat neutral pH values, including but not limited to, under physiologicalconditions for an extended period of time, perhaps even indefinitely.Hydrolytically unstable or degradable linkages mean that the linkagesare degradable in water or in aqueous solutions, including for example,blood. Enzymatically unstable or degradable linkages mean that thelinkage is often degraded by one or more enzymes. By way of example, PEGand related polymers include degradable linkages in the polymer backboneor in the linker group between the polymer backbone and one or more ofthe terminal functional groups of the polymer molecule. Such degradablelinkages include, but are not limited to, ester linkages formed by thereaction of PEG carboxylic acids or activated PEG carboxylic acids withalcohol groups on a biologically active agent, wherein such ester groupsgenerally hydrolyze under physiological conditions to release thebiologically active agent. Other hydrolytically degradable linkagesinclude but are not limited to carbonate linkages; imine linkagesresulted from reaction of an amine and an aldehyde; phosphate esterlinkages formed by reacting an alcohol with a phosphate group; hydrazonelinkages which are reaction product of a hydrazide and an aldehyde;acetal linkages that are the reaction product of an aldehyde and analcohol; orthoester linkages that are the reaction product of a formateand an alcohol; peptide linkages formed by an amine group, including butnot limited to, at an end of a polymer such as PEG, and a carboxyl groupof a peptide; and oligonucleotide linkages formed by a phosphoramiditegroup, including but not limited to, at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

The conjugates for use in the method described herein is conjugated byusing any art-recognized method forming a complex including covalent,ionic, or hydrogen bonding of the ligand to the imaging agent, eitherdirectly or indirectly via a linking group such as a linker. Theconjugate is typically formed by covalent bonding of the ligand to theimaging agent through the formation of amide, ester or imino bondsbetween acid, aldehyde, hydroxy, amino, or hydrazo groups on therespective components of the complex or, for example, by the formationof disulfide bonds.

In addition, structural modifications of a linker portion of theconjugates are contemplated herein. For example, a number of amino acidsubstitutions are often made to the linker portion of the conjugate,including but not limited to naturally occurring amino acids, as well asthose available from conventional synthetic methods. In one aspect,beta, gamma, and longer chain amino acids are used in place of one ormore alpha amino acids. In another aspect, the stereochemistry of thechiral centers found in such molecules is selected to form variousmixture of optical purity of the entire molecule, or only of a subset ofthe chiral centers present. In another aspect, the length of the peptidechain included in the linker is shortened or lengthened, either bychanging the number of amino acids included therein, or by includingmore or fewer beta, gamma, or longer chain amino acids. In anotheraspect, the selection of amino acid side chains in the peptide portionis made to increase or decrease the relative hydrophilicity of thelinker portion specifically or of the overall molecule generally.

Similarly, the length and shape of other chemical fragments of thelinkers described herein is often modified. In some aspects, the linkerincludes an alkylene chain. The alkylene chain often varies in length,or includes branched groups, or includes a cyclic portion, which are inline or spiro relative to the allylene chain. In another aspect, wherethe linker includes a beta thiol releasable fragment, it is appreciatedthat other intervening groups connecting the thiol end to the hydroxy orcarbonate end are used in place of the ethylene bridge, such as but notlimited to optionally substituted benzyl groups, where the hydroxy endis connected at the benzyl carbon and the thiol end is connected throughthe ortho or para phenyl position, and vice versa.

Formulations of Chlorotoxin Conjugates

In various aspects, the present disclosure provides compositionscomprising the above-described compounds and a pharmaceuticallyacceptable carrier. In some aspects, the composition is formulated forparenteral administration. In further aspects, the composition isformulated for intravenous administration, intramuscular administration,subcutaneous administration, or a combination thereof.

Certain methods described herein comprise administering to the subjectan intravenous pharmaceutical composition comprising a chlorotoxinconjugate, for example, as described herein. Intravenous pharmaceuticalcompositions of chlorotoxin conjugates include any formulation suitablefor administration to a subject via any intravenous method, including abolus, an infusion which occurs over time or any other intravenousmethod known in the art. In some aspects, the rate of infusion is suchthat the dose is administered over a period of less than five minutes,more than five minutes but less than 15 minutes or greater than 15minutes. In other aspects, the rate of infusion is such that the dose isadministered over a period of less than 5 minutes. In other aspects, therate of infusion is such that the dose is administered over a period ofgreater than 5 minutes and less than 15 minutes. In some other aspects,the rate of infusion is such that the dose is administered over a periodof greater than 15 minutes.

“Product” or “dosage form” as used herein refers to any solid,semi-solid, lyophilized, aqueous, liquid or frozen formulation orpreparation used for administration. Upon administration, the rate ofrelease of an active moiety from a product is often greatly influencedby the excipients and/or product characteristics which make up theproduct itself. For example, an enteric coat on a tablet is designed toseparate that tablet's contents from the stomach contents to prevent,for example, degradation of the stomach which often inducesgastrointestinal discomfort or injury. According to the currentlyaccepted conventional understanding, systemic exposure of the activemoiety will be relatively insensitive to the small formulation changes.

As used herein “pharmaceutically acceptable” or “pharmacologicallyacceptable” includes molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to a subject, as appropriate. “Pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients are often also incorporated into the compositions.

In various aspects, the present compositions comprise a concentration ofthe compound as an active pharmaceutical ingredient having aconcentration of from 1 mg/mL to 40 mg/mL. In further aspects, theconcentration of the compound is from 1 mg/mL to 20 mg/mL. In stillother aspects, the concentration of the compound is from 4 mg/mL to 10mg/mL. In additional aspects, the concentration of the compound is from5 mg/mL to 8 mg/mL. In yet further aspects, concentration of thecompound is from 5 mg/mL to 6 mg/mL.

In some aspects, pharmaceutically acceptable carrier comprises tris,D-mannitol, and a pH of essentially 6.8. In other aspects, thecompositions consist essentially of tris, D-mannitol, and a pH of 6.8.

In some aspects, pharmaceutically acceptable carrier comprises histidineand mannitol. In some aspects, pharmaceutically acceptable carriercomprises histidine and mannitol with polysorbate 20. In some aspects,pharmaceutically acceptable carrier comprises L-histidine, D-mannitol,L-methionine, and a pH of essentially 6.8. In additional aspects, thepharmaceutically acceptable carrier consists essentially of L-histidine,D-mannitol, L-methionine, and a pH of 6.8.

In some aspects, the pharmaceutically acceptable carrier comprisesL-histidine, D-mannitol, polysorbate 20, and a pH of essentially 6.8. Insome aspects, the pharmaceutically acceptable carrier comprisesL-histidine, D-mannitol, and a pH of essentially 6.8. In some aspects,the pharmaceutically acceptable carrier comprises L-histidine,D-mannitol, polysorbate 20, trehalose, and a pH of essentially 6.8.

A pharmaceutical composition comprising a chlorotoxin conjugate isformulated according to known methods to prepare pharmaceutically usefulcompositions, for example, as found in “Excipient Selection inParenteral Formulation Development” Pramanick et.al., Pharma Times, Vol.45., No. 3, Mar. 2013, incorporated in its entirety herein by reference.In some aspects, the chlorotoxin conjugate is combined with apharmaceutically acceptable carrier. A composition is said to be apharmaceutically acceptable carrier if its administration is toleratedby a recipient patient. Sterile phosphate-buffered saline is one exampleof a pharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

Formulations for administration of chlorotoxin conjugates are typicallyprovided but are not limited to as liquid, solid or semi-solid productsor dosage forms, exemplified by tablets, capsules, pellets, a powder ora lyophilized product. In some aspects, the chlorotoxin conjugate isformulated to comprise no additional materials except for apharmaceutical carrier. In some other aspects, the chlorotoxin conjugateis formulated such that it comprises a core “matrix material” whichencapsulates, binds to, coats or is adjacent to the chlorotoxinconjugate. In some other aspects, the chlorotoxin conjugate and matrixmaterial further comprises a protective coatings. Various formulationsare well-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

Suitable excipients for use with chlorotoxin conjugates are oftenincluded in formulations for intravenous use, for example, an injection.Injections are sterile, pyrogen-free solutions or dispersions (emulsionsor suspensions) of one or more active ingredients in a suitable vehicleor carrier. Injections that are dispersions should remain sufficientlystable so that, after shaking, a homogeneous dose is withdrawn. Morespecifically, formulations which include chlorotoxin conjugates and oneor more but not limited to suitable excipients, exemplified by matrixmaterials, binders, lubricants, glidants or disintegrants which aid inmodulating the PK profile of administered chlorotoxin conjugates arepreferred. In some aspects, compositions comprising chlorotoxinconjugates in combination with one or more suitable excipients and oneor more specific product characteristics (such as dissolution or watercontent) which result in improved pharmacokinetic profiles ofchlorotoxin conjugates in vivo. Thus, the in vivo performance ofchlorotoxin conjugates dosage forms/products included herein is basedupon the composition of the excipients added during manufacturing and/orthe final product characteristics generated through specific processingparameters and methods. Other excipients are well-known to those in theart. See, for example, Gennaro (ed.), Remington's PharmaceuticalSciences, 19th Edition (Mack Publishing Company 1995).

Suitable carriers for intravenous administration include for example butare not limited to physiological saline or phosphate buffered saline(PBS), Tris, and solutions containing solubilizing agents, such asglucose, polyethylene glycol, polypropylene glycol, additional agentssuch as histidine, dextrose, mannitol and mixtures thereof. In someaspects, carriers for intravenous administration include a mixture ofhistidine and dextrose, Tris and dextrose or Tris and mannitol. Othercarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

The formulation often includes an aqueous vehicle. Aqueous vehiclesinclude, by way of example and without limitation, sodium chloridesolution, Ringers solution, isotonic dextrose solution, sterile watersolution, dextrose and lactated Ringers solution. Nonaqueous vehiclesinclude, by way of example and without limitation, fixed oils ofvegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil,benzyl benzoate, castor oil, N,N-dimethylacetamide, ethanol, dehydratedethanol, glycerin, glycerol, N-methyl-2-pyrrolidone, polyethylene glycoland any derivative thereof, propylene glycol, safflower oil and soybeanoil. Other vehicles are well-known to those in the art. See, forexample, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19thEdition (Mack Publishing Company 1995).

In some aspects, the composition the pharmaceutically acceptable carriercomprises an osmolyte. In some aspects, the osmolyte comprises a sugar,a sugar alcohol, or a combination thereof

In certain aspects, the composition comprises a sugar alcohol selectedfrom sorbitol, inositol, mannitol, xylitol and glycerol, or acombination thereof. In further aspects, the sugar alcohol comprisesmannitol. In certain aspects, the composition comprises from 2% to 20%(wt/vol %) mannitol. In some aspects, the composition comprises from 2%to 10% (wt/vol %) mannitol. In further aspects, the compositioncomprises essentially 5% (wt/vol %) mannitol.

In other aspects, the composition comprises a sugar. In certain aspects,the sugar is selected from trehalose, lactose, sucrose, glucose,galactose, maltose, mannose, fructose, dextrose, or a combinationthereof. In additional aspects, the sugar is selected from trehalose,sucrose, or a combination thereof. In some aspects, the compositioncomprises from 1% to 40% (wt/vol %) of trehalose, sucrose, or acombination of trehalose and sucrose. In other aspects, the compositioncomprises from 1% to 20% (wt/vol %) of trehalose, sucrose, or acombination of trehalose and sucrose. In additional aspects, thecomposition comprises 2% (wt/vol %) of trehalose, sucrose, or acombination of trehalose and sucrose.

In certain aspects, the composition further comprises an osmolyteselected from glycine, carnitine, ethanolamine, their phosphates, monosugars, or a combination thereof.

In some aspects, the present compositions are isotonic. In otheraspects, the compositions are essentially isotonic.

In certain aspects, the ionic strength of the composition is less than50 mM. In other aspects, the ionic strength of the composition is lessthan 10 mM.

Antimicrobial agents in bacteriostatic or fungistatic concentrations aretypically added to preparations packaged in multiple dose containerswhich include by way of example and without limitation, phenols orcresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Other antimicrobial agents are well-known tothose in the art. See, for example, Gennaro (ed.), Remington'sPharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).

Buffers include by way of example and without limitation, acetate,ammonium sulfate, ammonium hydroxide, arginine, aspartic acid, benzenesulfonic acid, benzoate sodium, benzoate acid, carbonate, sodiumcarbonate, carbon dioxide, citrate, diethanolamine, glucono deltalactone, glycine, glycine HC1, histidine, histidine HC1, hydrochloricacid, hydrobromic acid, lysine maleic acid, meglumine, methanesulfonicacid, monoethanolamine, phosphate, sodium phosphate, citrate, succinatesodium, sulfuric acid, tartarate sodium, trmethamine, sodium citrate,hydroxide, sodium hydroxide, Tris base, Tris base -65, Tris acetate,Tris HCl, and Tris HC1-65.

In various aspects, the pharmaceutically acceptable carrier comprises abuffer. In some aspects, the buffer is selected from tris, HEPES,histidine, ethylene diamine, or a combination thereof. In other aspects,the buffer is selected from tris, histidine, or a combination thereof.In further aspects, the buffer comprises histidine, which is optionallyL-histidine. In additional aspects, the composition comprises at least100 mM histidine. In further aspects, the composition comprises at least50 mM histidine. In some aspects, the composition comprises at least 20mM histidine. In additional aspects, the composition comprises 10 to 100mM histidine. In other aspects, the composition comprises 10 to 20 mMhistidine.

Antioxidants include by way of example and without limitation, sodiumbisulfate, acetone sodium bisulfate, argon, ascorbyl palmitate,ascorbate sodium, ascorbate acid, butylated hydroxy anisole, butylatedhydroxy toluene, cysteine, cystenate HC1, dithionite sodium, gentisticacid, gentistic acid ethanoloamine, glutamate monosodium, glutathione,formaldehyde solfoxylate sodium, metabisulfite potassium, metabisulfitesodium, methionine, monothioglycerol, nitrogen, propyl gallate, sulfitesodium, tocopherol alpha, alpha tocopherol hydrogen succinate andthioglycolyate sodium.

In some aspects, the compositions comprise an antioxidant, a freeradical scavenger, a quencher, an antioxidant synergist or a combinationthereof.

In some aspects, the antioxidant is selected from methionine, butylatedhydroxytoluene, butylated hydroxyanisole, propyl gallate, or acombination thereof. In other aspects, the antioxidant comprisesmethionine. In further aspects, the antioxidant is L-methionine. Incertain aspects, the compositions comprise at least 20 mM methionine. Inother aspects, aspects, the compositions comprise at least 10 mMmethionine.

Suspending, emulsifying and/or dispersing agents include by way ofexample and without limitation, sodium carboxymethylcelluose,hydroxypropyl methylcellulose, Polysorbate 80 (TWEEN® 80) andpolyvinylpyrrolidone.

In various aspects, the compositions comprise a surfactant. In certainaspects, the surfactant is selected from polysorbate 20, polysorbate 80,a pluronic, polyoxyethylene sorbitan mono-oleate, polyethylenemono-laureate, N-actylglucoside, or a combination thereof. In certainaspects, the surfactant is polysorbate 20. In further aspects, thecompositions comprise from 0.0001% to 0.1% (wt/vol %) polysorbate 20. Inadditional aspects, the compositions comprise cyclodextrin. In furtheraspects, the cyclodextrin comprises (2-hydroxypropyl)-β-cyclodextrin.

A sequestering or chelating agent of metal ions include by way ofexample and without limitation, calcium disodium EDTA, disodium EDTA,sodium EDTA, calcium versetaminde sodium, calteridol and DPTA. In someaspects, the present compositions comprise a metal chelator. In certainaspects, the metal chelator is selected from EDTA, deferoxaminemesylate, EGTA, fumaric acid, and malic acid, salts thereof, orcombinations thereof. In further aspects, the metal chelator comprisesEDTA or salts thereof. In certain aspects, the compositions have an EDTAconcentration of about 0.1 mg/ml to about 1.0 mg/ml.

Other isotonic agents, buffers, antioxidants, anesthetics, suspendingand dispersing agents, emulsifying agents and chelating agents arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

Pharmaceutical carriers also include, by way of example and withoutlimitation, ethyl alcohol, polyethylene glycol and propylene glycol forwater miscible vehicles and sodium hydroxide, hydrochloric acid, citricacid or lactic acid. Other pharmaceutical carriers are well-known tothose in the art. See, for example, Gennaro (ed.), Remington'sPharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).

The chlorotoxin conjugates described herein are often formulated using avariety of parameters including by way of example and withoutlimitation, pH, molarity, % weight/volume, % volume/volume and the like.Other factors considered in the formulation of, stability of, storageof, shipping of chlorotoxin conjugates include by way of example andwithout limitation, the gas environment, container material, containercolor, cap material, cap color, presence of additional aspects, such asantioxidants, stabilizers, photoprotective compounds, protectants,sugars, ion chelators, ion donors or the like. Any factor which servesas any one of the above factors known to one of ordinary skill in theart is often used with the chlorotoxin conjugates described herein butnot limited as such.

The preparation of pharmaceutical or pharmacological compositions areknown to those of skill in the art in light of the present disclosure.General techniques for formulation and administration are found in“Remington: The Science and Practice of Pharmacy, Twentieth Edition,”Lippincott Williams & Wilkins, Philadelphia, Pa. Tablets, capsules,pills, powders, granules, dragees, gels, slurries, ointments, solutionssuppositories, injections, inhalants and aerosols are examples of suchformulations.

The chlorotoxin conjugates are often stored at various temperatures,including by way of example and without limitation, freezing, forexample at about −20° C., about −70° C., about −100° C., about −120° C.,about −150° C., about −200° C. or more than about −200° C., coldstorage, for example at about 10° C., about 5° C., about 4° C., about 2°C., about 0° C., about −2° C. or more than about −5° C., or any othersuitable temperature such that the composition remains stable.

In some aspects, compositions comprising the compounds described hereinare stored as lyophilized solids. In some aspects, the presentdisclosure provides methods for producing the lyophilized composition,the method comprising providing the composition; and lyophilizing thecomposition, thereby producing the lyophilized composition.

Using lyophilization, it is possible to store the compounds in a mannerthat maintains physiological or otherwise optimal pH, isotonicity andstability. Such materials include pH buffers, preservatives, tonicityadjusting agents, anti-oxidants, other polymers (e.g., viscosityadjusting agents or extenders) and excipients to stabilize the labileprotein against the stresses of drying and storage of the dried product.Specific illustrative examples of such additives include phosphate,citrate, or borate buffers; thimerosal; sorbic acid; methyl or propylparaben, and chlorobutanol preservatives; sodium chloride: polyvinylalcohol, polyvinyl pyrrolidone; mannitol, dextrose, dextran, lactose,sucrose, ethylene diamine tetra-acetic acid, and the like. Suitableformulations, known in the art, (Remington's Pharmaceutical Sciences(latest edition), Mack Publishing Company, Easton, Pa.; Arakawa et al.(1990), supra; Carpenter et al. (1991), supra; and Pikal (1990), supra).

In certain aspects, the pharmaceutically acceptable carrier comprises areconstitution stabilizer. In other aspects, the reconstitutionstabilizer comprises a water-soluble polymer. In additional aspets, thewater-soluble polymer is selected from a polaxamer, a polyol, apolyethylene glycol, a polyvinylalcohol, a hydroxyethyl starch, dextran,polyvinylpyrrolidene poly(acrylic acid), or a combination thereof.

The term “reconstitution stabilizer” means any excipient which iscapable of preventing aggregation of a reconstituted protein in anaqueous medium. Excipients possessing the necessary characteristics forthe present invention are well-known in the art and generally functionby the mechanisms of charge replusion, steric hindrance, hydrophobicbinding or specific high-affinity binding to the dried protein.Exemplary excipients include various osmolytes, various salts, watersoluble synthetic and natural polymers, surfactants, sulfatedpolysaccharides, carrier proteins, buffers and the like (Manning et al.(1989), Pharmaceutical Research, 6:903-918; and Paborji, et al. (1994),Pharmaceutical Research, 11:764-771).

The present compounds and an effective amount of the reconstitutionstabilizer are admixed under conditions effective to reduce aggregationof present compounds upon reconstitution with the reconstitution medium(e.g., a solvent and optionally other components such asantibacterials). The reconstitution stabilizer may be admixed with thecompouns at a suitable time before, during or after reconstitution;preferably the reconstitution stabilizer will be pre-dissolved in thereconstitution medium. The compound is reconstituted at a temperaturewhich is above the freezing point of the reconstitution medium, butwhich will not degrade the compound and which will not be deleterious tothe reconstitution stabilizer; preferably the temperature will bebetween about 2° C. to 50° C. The time taken to mix the reconstitutionstabilizer and the dried compound should be for a sufficient period toprepare a suitable admixture; preferably mixing will be for betweenabout 1 to 30 minutes. Generally, the reconstituted formulation is usedsoon after reconstitution.

In certain aspects, the present compositions are reconstituted from alyophilized form. In other aspects, the present disclosure providesmethods for producing the reconstituted composition, the methodcomprising providing a lyophilized composition; and reconstituting thecomposition with a solution to produce a reconstituted composition. Invarious aspects, the reconstituting solution comprises water. In someaspects, the reconstituting solution is selected from sterile water,physiological saline solution, glucose solution or other aqueoussolvents (e.g., alcohols such as ethyl, n-propyl or isopropyl, butylalcohol), or a combination thereof, which are capable of dissolving thedried composition and compatible with the selected administration routeand which does not negatively interfere with the compound and thereconstitution stabilizers employed.

Storage Vessels

In some aspects, the chlorotoxin conjugates are placed into containersfollowing formulation. Often the containers include by way of exampleand without limitation, glass, for example amber glass or colorlessglass. The containers often include by way of example and withoutlimitation, a plastic, a rubber, a metal, a biodegradable material orthe like known to one of skill in the art and are any color, colorless,opaque or clear. In some aspects, the chlorotoxin conjugates are placedinto containers which are sealed following formulation. Often the sealis a cap, for example, the cap is a plastic, a rubber, a metal, abiodegradable material, a combination of or the like known to one ofskill in the art and are any color, colorless, opaque or clear,sometimes a film. In some aspects, a gas is included in the container,often to enhance stability of the chlorotoxin conjugate or preventoxygen from contacting the chlorotoxin conjugate. For example, gasincludes by way of example and without limitation, an inert gas, such asnitrogen or argon, occasionally a noble gas and is used at any suitableconcentration.

In some aspects the compounds of the present disclosure are stored in avessel comprising glass, particularly a Type I glass, which has beensubjected to a washing or extraction treatment which reduces the levelof extractable trivalent and divalent metal ions present in/on thesurface of the glass. Such treatments include steeping in (extractionwith) hot (preferably at least 90° C.) water or another aqueous medium,e.g. ammonium sulfate solution, or treatment with sulfur dioxide.

In some aspects, the compounds of the present disclosure are stored in avessel comprising USP Type 1 borosilicate glass vial with a 13 mmchlorobutyl based stopper with flourotech coating on plug and B2 coatingon the top and an aluminum over seal with flip top cap.

In certain aspects, the compounds of the present disclosure are storedin a foil-lined chamber. In some aspects, the chamber comprises a dryinert atmosphere, preferably nitrogen, and a desiccant or oxygenabsorber.

In various aspects, the present disclosure provides a kit comprisingvessel configured to contain a fluid; any of the compounds andcompositions described herein; and an elastomeric closure affixed to thevessel.

In some aspects, the kit further comprises a light shield. In furtheraspects, the light shield is a physical barrier configured to block atleast a portion of the light incident on the vessel from thecomposition. In still further aspects, the physical barrier comprises anopaque or semi-opaque material.

In some aspects, the vessel is a glass vial. In further aspects, theglass vial comprises clear or amber glass. In some aspects, the glassvial is an untreated glass container. In certain aspects, the glass vialcomprises USP Type I, Type II, Type III, or Type IV glass. In someaspects, the inner portion of the vessel further comprises a silica(SiO₂) coating or silicone coating. In some aspects, the untreated glasscontainer is selected from an ampoule, vial, ready-to-use syringe, orcarpoule.

In some aspects, the elastomeric closure is a halobutyl rubber closure.In further aspects, the halobutyl rubber closure is selected from achlorobutyl rubber closure or a bromobutyl rubber closure. In someaspects, elastomeric closure is coated with Fluorotec, B2, or acombination thereof.

In some aspects, the kit further comprises an opaque secondary packagesurrounding the vessel. In certain aspects, the opaque secondary packagecomprises an opaque box, an opaque aluminum foil pouch, or a combinationthereof. In further aspects, the opaque secondary package is configuredto block at least 90% of the light incident on the package exterior fromthe composition. In still further aspects, the opaque secondary packageis configured to block at least 95% of the light incident on the packageexterior from the composition. In still further aspects, the opaquesecondary package is configured to block at least 99% of the lightincident on the package exterior from the composition. In still furtheraspects, the opaque secondary package is configured to block at least99.9% of the light incident on the package exterior from thecomposition.

In some aspects, the vessel comprises a reduced-oxygen environment incontact with the composition. In certain aspects, the vessel comprisesan inert gas in contact with the composition. In some aspects, thecomposition is sparged with an inert gas, thereby producing thereduced-oxygen environment in the vessel. In certain aspects, the inertgas comprises nitrogen or argon.

Dosages and Toxicity of Compounds

The product or dosage form characteristics which result from theprocessing methods and/or parameters for generating formulations such aspowders, lyophilized compositions, and the like include, but are notlimited to, density, water content, friability, disintegration,dissolution profile(s), shape, size, weight, uniformity and compositionof the particles. These product characteristics are often modulated in anumber of ways and affect the final in vitro and/or in vivo performanceof the formulations. Product or dosage form characteristics are often aconsequence of excipient selection, excipient composition, manufacturingmethods applied or a combination of any of these. The combination ofexcipients as well as product characteristics (including processingmethods or processing parameters) of the final dosage form willultimately determine the pharmacokinetic profile of the activeingredient in vivo. The administered chlorotoxin conjugate formulationsdescribed herein are often processed or manufactured under specificconditions such as, for example, mixing methods (including sieve size,rpm, and milling), drying time, conditions, environmental parameters(e.g., temperature and humidity) and combinations thereof) whichthemselves modulate the pharmacokinetic profile of chlorotoxincompositions in vivo (i.e., increase the average C_(max) or AUC). Inorder to quantitatively compare one formulation to another, it iscustomary to measure several of these product or dosage formcharacteristics. This is also necessary when attempting to duplicatemultiple batches.

Dissolution and drug release from formulations depends on many factorsincluding the solubility and concentration of the active ingredient, thenature and composition of the excipients, content uniformity, watercontent, product shape and size, porosity, disintegration time and otherfactors. The release of a drug or active ingredient from a final dosageform in vitro is typically characterized by its dissolution profileunder standardized conditions (using United States Pharmacopeia (USP) orsimilar accepted methods for reference) and at the appropriate pH, oftena neutral pH. The dissolution profile shows the amount of drug releasedover time into the test media under specified conditions. Standardconditions make use of buffers at an appropriate pH in order to bestmimic the pH of a subject's blood.

Typically a therapeutically effective dosage is formulated to contain adose of at least about 0.1 mg up to about 1.5 mg or more, such as morethan 1.5 mg of chlorotoxin conjugate. In some aspects, the effectivedosage is formulated to contain a dose of at least about 0.01 mg, about0.02 mg, about 0.03 mg, about 0.5 mg, about 0.07 mg, about 0.1 mg, about0.2 mg, about 0.3 mg, about 0.35 mg, about 0.375 mg, about 0.4 mg, about0.5 mg, about 0.6 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about0.9 mg, about 1 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.8mg, about 1.9 mg, about 2 mg, about 2.4 mg, about 3 mg, about 5 mg,about 6 mg, about 7 mg, about 12 mg, about 18 mg or about 60 mg more ofchlorotoxin conjugate. In an exemplary aspect, the dose is 0.1 mg for amouse, 1 mg for a dog, 0.3 mg for a rat, 0.6 mg for a monkey and 3 mgfor a human. The amount of chlorotoxin conjugate administered to asubject is often the total about amount listed herein. In some aspects,the amount of chlorotoxin conjugate administered to a subject is oftenthe about per milligram, gram or kilogram of subject weight for eachamount listed herein. In other aspects, the amount of chlorotoxinconjugate administered to a subject is often the about per milliliter orliter of fluid volume for each amount listed herein. In yet otheraspects, the amount of chlorotoxin conjugate administered to a subjectis often the about per square millimeter, square centimeter or squaremeter of subject surface body area or subject body area for each amountlisted herein.

As used herein a “dosage regimen” refers to the protocol used toadminister an intravenous pharmaceutical formulation comprisingchlorotoxin conjugate to a subject. In some aspects, the dosage regimencomprises a dose amount and dosing interval. In some aspects, the dosageregimen further comprises a dosing duration. As used herein “dosingduration” refers to the period of time over which a dose isadministered.

In some aspects, the dose of chlorotoxin conjugate is administered to asubject using either a fixed or a scaling dosing scheme. For example, afixed dosing scheme includes administration of a bolus or continuousdose of chlorotoxin conjugate to a subject via an intravenousadministration route wherein the fixed dose is, for example and withoutlimitation, about 3 mg to about 6 mg and does not account or adjust fora subject's age, weight, height, body mass index, metabolism, or thelike. For example, a scaling dosing scheme includes administration of abolus or continuous dose of chlorotoxin conjugate to a subject via anintravenous administration route wherein the scaled dose is, for exampleand without limitation, about 3 mg to about 6 mg and accounts or adjustsfor a subject's age, weight, height, body mass index, metabolism, or thelike. In some aspects, the fixed dose and/or the scaled dose aredetermined for one subject based upon the dose administered to adifferent subject wherein the subjects are or are not the same species,for example a mouse and a human or a rat and a human, or a dog and ahuman or a monkey and a human or a non-human primate and a human. Oftenin a fixed dose, the same dose or about the same dose is administered toall subjects, for example a mouse and a human or a rat and a human, or adog and a human or a monkey and a human or a non-human primate and ahuman. In some aspects, the scaled dose to be administered to a subjectis determined from the dose administered to a different subject whereinthe subjects are or are not the same species, for example a mouse and ahuman or a rat and a human, or a dog and a human or a monkey and a humanor a non-human primate and a human. The scaled dose is thereforeincreased from the dose administered to the mouse, rat, dog, monkey ornon-human primate to the dose administered to the human based upon thedifference between the mouse, rat, dog, monkey or non-human primate andthe human on factors such as subject age, weight, height, metabolism,size or the like. In a preferred aspect, the dose is scaled from a ratto a human.

The compounds and compositions described herein are administered to asubject before surgery, during surgery and/or the excised tissue fromthe subject is contacted with compositions of the chlorotoxinconjugates. In some aspects, compositions of chlorotoxin conjugates areintravenously administered to a subject about 1 hour, about 2 hours,about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 9hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours orabout 72 hours before surgery. In some aspects, compositions ofchlorotoxin conjugates are intravenously administered to a subjectbetween 0 and 1 hours, between 1 and 2 hours, between 2 and 3 hours,between 3 and 4 hours, between 4 and 5 hours, between 5 and 6 hours,between 6 and 9 hours, between 9 and 12 hours, between 12 and 24 hours,between 24 and 36 hours, between 36 and 48 hours or between 48 and 72hours (inclusive) before surgery. Tissue or fluid samples are oftenisolated from a subject prior to administration of a chlorotoxinconjugate, sometimes as a baseline reference. Samples are also isolatedfrom a subject after administration of the compounds of the presentdisclosure, often less than about 1 minute after, about 2 minutes after,about 3 minutes after, about 4 minutes after, about 5 minutes after,about 6 minutes after, about 7 minutes after, about 8 minutes after,about 9 minutes after, about 10 minutes after, about 11 minutes after,about 12 minutes after, about 13 minutes after, about 14 minutes after,about 15 minutes after, about 20 minutes after, about 30 minutes after,about 40 minutes after, about 50 minutes after, about 60 minutes after,about 1 hour after, about 2 hours after, about 3 hours after, about 4hours after, about 5 hours after, about 6 hours after, about 12 hoursafter, about 18 hours after, about 24 hours after, about 36 hours after,about 48 hours after, about 72 hours after, about 96 hours after, about5 days after, about 7 days after, about 10 days after, about 14 daysafter, about 21 days after, about 4 weeks after, about 6 weeks after,about 8 weeks after, about 12 weeks after, about 16 weeks after, about20 weeks after or more than 20 weeks after.

Pharmacokinetics

The methods and compositions described herein relate to pharmacokineticsof intravenous administration of chlorotoxin conjugates to a subject.Pharmacokinetics are often described using models, for example,compartmental or noncompartmental models. Compartmental models includebut are not limited to monocompartmental model, the two compartmentalmodel, the multicompartmental model or the like. Models are oftendivided into different compartments and described by the correspondingscheme. For example, one scheme is the absorption, distribution,metabolism and excretion (ADME) scheme. For another example, anotherscheme is the liberation, absorption, distribution, metabolism andexcretion (LADME) scheme. In some aspects, metabolism and excretion aregrouped into one compartment referred to as the elimination compartment.For example, liberation includes liberation of the active portion of thecomposition from the delivery system, absorption includes absorption ofthe active portion of the composition by the subject, distributionincludes distribution of the composition through the blood plasma and todifferent tissues, metabolism, which includes metabolism or inactivationof the composition and finally excretion, which includes excretion orelimination of the composition or the products of metabolism of thecomposition. Often, compositions administered intravenously to a subjectare subject to multiphasic absorption including but not limited toaspects of tissue distribution and metabolism/excretion. As such, thedecrease in plasma concentration of the composition is often biphasic,including, for example an alpha phase and a beta phase, occasionally agamma, delta or other phase is observed. In some aspects, thebioavailability of the compositions described herein is absolutebioavailability, often 1 or 100% given intravenous administration.

Pharmacokinetics includes determining at least one parameter associatedwith intravenous administration of chlorotoxin conjugates to a subject.In some aspects, parameters include at least the dose (D), dosinginterval (τ), area under curve (AUC), maximum concentration (C_(max)),minimum concentration reached before a subsequent dose is administered(C_(min)), minimum time (T_(min)), maximum time to reach C_(max)(T_(max)), volume of distribution (V_(d)), back-extrapolatedconcentration at time 0 (C₀), steady state concentration (C_(ss)),elimination rate constant (k_(e)), infusion rate (k_(in)), clearance(CL), bioavailability (f), fluctuation (% PTF) and elimination half-life(T_(1/2)). In another aspect, the C_(max), C₀. and AUC increase in adose-dependent manner.

The compounds described herein have values for at least one of thepharmacokinetic parameters listed herein and known to those of ordinaryskill in the art. Often, the values for the pharmacokinetic parametersare recorded, observed, measured, processed, analyzed or the like asdata. The pharmacokinetics parameters are any parameters suitable fordescribing the plasma or serum profiles of chlorotoxin conjugatesdescribed herein. For example, the pharmacokinetics profile are oftenobtained at a time after dosing of, for example, about zero minutes,about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57minutes, about 58 minutes, about 59 minutes, about 60 minutes, aboutzero hours, about 0.5 hours, about 1 hour, about 1.5 hours, about 2hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours,about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours,about 11 hours, about 11.5 hours, about 12 hours, about 12.5 hours,about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours,about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours,about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours,about 19 hours, about 19.5 hours, about 20 hours, about 20.5 hours,about 21 hours, about 21.5 hours, about 22 hours, about 22.5 hours,about 23 hours, about 23.5 hours, or about 24 hours.

The pharmacokinetics parameters are any parameters suitable fordescribing the plasma or serum profiles of chlorotoxin conjugatesdescribed herein. In some aspects, the dose (D) includes by way ofexample but is not limited to, about 0.01 mg, about 0.02 mg, about 0.03mg, about 0.5 mg, about 0.07 mg, about 0.1 mg, about 0.2 mg, about 0.3mg, about 0.35 mg, about 0.375 mg, about 0.4 mg, about 0.5 mg, about 0.6mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.9 mg, about 1 mg,about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.8 mg, about 1.9 mg,about 2 mg, about 2.4 mg, about 3 mg, about 5 mg, about 6 mg, about 7mg, about 12 mg, about 18 mg or about 60 mg more of chlorotoxinconjugate. In some aspects, the dosing interval (τ) includes by way ofexample but is not limited to, about 12 hours, about 24 hours, about 36hours, about 48 hours or about 72 hours before surgery.

The pharmacokinetics parameters are any parameters suitable fordescribing the plasma or serum profiles of chlorotoxin conjugatesdescribed herein. In some aspects, the area under curve (AUC) includesby way of example but is not limited to, not less than about 50hr*ng/mL, not less than about 75 hr*ng/mL, not less than about 100hr*ng/mL, not less than about 125 hr*ng/mL, not less than about 150hr*ng/mL, not less than about 175 hr*ng/mL, not less than about 200hr*ng/mL, not less than about 250 hr*ng/mL, not less than about 300hr*ng/mL, not less than about 350 hr*ng/mL, not less than about 400hr*ng/mL, not less than about 500 hr*ng/mL, not less than about 600hr*ng/mL, not less than about 700 hr*ng/mL, not less than about 800hr*ng/mL, not less than about 900 hr*ng/mL, not less than about 1000hr*ng/mL, not less than about 2000 hr*ng/mL, not less than about 3000hr*ng/mL, not less than about 4000 hr*ng/mL, not less than about 5000hr*ng/mL, not less than about 6000 hr*ng/mL, not less than about 7000hr*ng/mL, not less than about 8000 hr*ng/mL, not less than about 9000hr*ng/mL, not less than about 10000 hr*ng/mL, not less than about 11000hr*ng/mL, not less than about 12000 hr*ng/mL, not less than about 13000hr*ng/mL, not less than about 14000 hr*ng/mL, not less than about 15000hr*ng/mL, not less than about 16000 hr*ng/mL, not less than about 17000hr*ng/mL, not less than about 18000 hr*ng/mL, not less than about 19000hr*ng/mL, not less than about 20000 hr*ng/mL, not less than about 21000hr*ng/mL, not less than about 22000 hr*ng/mL, not less than about 23000hr*ng/mL, not less than about 24000 hr*ng/mL, not less than about 25000hr*ng/mL, not less than about 26000 hr*ng/mL, not less than about 27000hr*ng/mL, not less than about 28000 hr*ng/mL, not less than about 29000hr*ng/mL, not less than about 30000 hr*ng/mL, not less than about 31000hr*ng/mL, not less than about 32000 hr*ng/mL, not less than about 33000hr*ng/mL, not less than about 34000 hr*ng/mL, not less than about 35000hr*ng/mL, not less than about 40000 hr*ng/mL not less than about 45000hr*ng/mL not less than about 50000 hr*ng/mL not less than about 55000hr*ng/mL not less than about 60000 hr*ng/mL not less than about 65000hr*ng/mL not less than about 70000 hr*ng/mL not less than about 75000hr*ng/mL not less than about 80000 hr*ng/mL not less than about 85000hr*ng/mL not less than about 90000 hr*ng/mL not less than about 95000hr*ng/mL not less than about 100000 hr*ng/mL not less than about 125000hr*ng/mL not less than about 150000 hr*ng/mL not less than about 175000hr*ng/mL not less than about 200000 hr*ng/mL not less than about 250000hr*ng/mL not less than about 300000 hr*ng/mL not less than about 350000hr*ng/mL not less than about 400000 hr*ng/mL not less than about 450000hr*ng/mL not less than about 500000 hr*ng/mL not less than about 550000hr*ng/mL not less than about 600000 hr*ng/mL not less than about 650000hr*ng/mL not less than about 700000 hr*ng/mL not less than about 750000hr*ng/mL not less than about 800000 hr*ng/mL not less than about 850000hr*ng/mL not less than about 900000 hr*ng/mL not less than about 950000hr*ng/mL not less than about 1000000 hr*ng/mL not less than about1100000 hr*ng/mL not less than about 1200000 hr*ng/mL not less thanabout 1300000 hr*ng/mL not less than about 1400000 hr*ng/mL not lessthan about 1500000 hr*ng/mL not less than about 1600000 hr*ng/mL notless than about 1700000 hr*ng/mL not less than about 1800000 hr*ng/mLnot less than about 1900000 hr*ng/mL not less than about 2000000hr*ng/mL or any other AUC appropriate for describing a pharmacokineticprofile of a chlorotoxin conjugate described herein.

The AUC of a chlorotoxin described herein by way of example can be, butis not limited to, about 1,000 hr*ng/mL to about 1,250 hr*ng/mL; about1,250 hr*ng/mL to about 1,500 hr*ng/mL; about 1,500 hr*ng/mL to about1,750 hr*ng/mL; about 1,750 hr*ng/mL to about 2,000 hr*ng/mL; about2,000 hr*ng/mL to about 2,500 hr*ng/mL; about 2,500 hr*ng/mL to about3,000 hr*ng/mL; about 3,000 hr*ng/mL to about 3,500 hr*ng/mL; about3,500 hr*ng/mL to about 4,000 hr*ng/mL; about 4,000 hr*ng/mL to about4,500 hr*ng/mL; about 4,500 hr*ng/mL to about 5,000 hr*ng/mL; about5,000 hr*ng/mL to about 5,500 hr*ng/mL; about 5,500 hr*ng/mL to about6,000 hr*ng/mL; about 6,000 hr*ng/mL to about 6,500 hr*ng/mL; about6,500 hr*ng/mL to about 7,000 hr*ng/mL; about 7,000 hr*ng/mL to about7,500 hr*ng/mL; about 7,500 hr*ng/mL to about 8,000 hr*ng/mL; about8,000 hr*ng/mL to about 8,500 hr*ng/mL; about 8,500 hr*ng/mL to about9,000 hr*ng/mL; about 9,000 hr*ng/mL to about 9,500 hr*ng/mL; about9,500 hr*ng/mL to about 10,000 hr*ng/mL; about 10,000 hr*ng/mL to about20,000 hr*ng/mL; about 20,000 hr*ng/mL to about 30,000 hr*ng/mL; about30,000 hr*ng/mL to about 40,000 hr*ng/mL; about 40,000 hr*ng/mL to about50,000 hr*ng/mL; about 50,000 hr*ng/mL to about 60,000 hr*ng/mL; about60,000 hr*ng/mL to about 70,000 hr*ng/mL; about 70,000 hr*ng/mL to about80,000 hr*ng/mL; about 80,000 hr*ng/mL to about 90,000 hr*ng/mL; about90,000 hr*ng/mL to about 100,000 hr*ng/mL; about 100,000 hr*ng/mL toabout 150,000 hr*ng/mL; about 150,000 hr*ng/mL to about 200,000hr*ng/mL; about 200,000 hr*ng/mL to about 250,000 hr*ng/mL; about250,000 hr*ng/mL to about 300,000 hr*ng/mL; about 300,000 hr*ng/mL toabout 350,000 hr*ng/mL; about 350,000 hr*ng/mL to about 400,000hr*ng/mL; about 400,000 hr*ng/mL to about 450,000 hr*ng/mL; about450,000 hr*ng/mL to about 500,000 hr*ng/mL; about 500,000 hr*ng/mL toabout 550,000 hr*ng/mL; about 550,000 hr*ng/mL to about 600,000hr*ng/mL; about 600,000 hr*ng/mL to about 650,000 hr*ng/mL; about650,000 hr*ng/mL to about 700,000 hr*ng/mL; about 700,000 hr*ng/mL toabout 750,000 hr*ng/mL; about 750,000 hr*ng/mL to about 800,000hr*ng/mL; about 800,000 hr*ng/mL to about 850,000 hr*ng/mL; about850,000 hr*ng/mL to about 900,000 hr*ng/mL; about 900,000 hr*ng/mL toabout 950,000 hr*ng/mL; about 950,000 hr*ng/mL to about 1,000,000hr*ng/mL; about 1,000,000 hr*ng/mL to about 1,100,000 hr*ng/mL; about1,100,000 hr*ng/mL to about 1,200,000 hr*ng/mL; about 1,200,000 hr*ng/mLto about 1,300,000 hr*ng/mL; about 1,300,000 hr*ng/mL to about 1,400,000hr*ng/mL; about 1,40,000 hr*ng/mL to about 1,500,000 hr*ng/mL; or about1,50,000 hr*ng/mL to about 2,000,000 hr*ng/mL.

The pharmacokinetic parameters is any parameters suitable for describinga chlorotoxin conjugate described herein. The C_(max) includes by way ofexample but is not limited to not less than about 1 ng/mL; not less thanabout 5 ng/mL; not less than about 10 ng/mL; not less than about 15ng/mL; not less than about 20 ng/mL; not less than about 25 ng/mL; notless than about 50 ng/mL; not less than about 75 ng/mL; not less thanabout 100 ng/mL; not less than about 200 ng/mL; not less than about 300ng/mL; not less than about 400 ng/mL; not less than about 500 ng/mL; notless than about 600 ng/mL; not less than about 700 ng/mL; not less thanabout 800 ng/mL; not less than about 900 ng/mL; not less than about 1000ng/mL; not less than about 1250 ng/mL; not less than about 1500 ng/mL;not less than about 1750 ng/mL; not less than about 2000 ng/mL; not lessthan about 2100 ng/mL; not less than about 2200 ng/mL; not less thanabout 2300 ng/mL; not less than about 2400 ng/mL; not less than about2500 ng/mL; not less than about 2600 ng/mL; not less than about 2700ng/mL; not less than about 2800 ng/mL; not less than about 2900 ng/mL;not less than about 3000 ng/mL; not less than about 3100 ng/mL; not lessthan about 32000 ng/mL; not less than about 3300 ng/mL; not less thanabout 3400 ng/mL; not less than about 3500 ng/mL; not less than about3600 ng/mL; not less than about 3700 ng/mL; not less than about 3800ng/mL; not less than about 3900 ng/mL; not less than about 4000 ng/mL;not less than about 4500 ng/mL; not less than about 5000 ng/mL; not lessthan about 5500 ng/mL; not less than about 6000 ng/mL; not less thanabout 6500 ng/mL; not less than about 2700 ng/mL; not less than about7500 ng/mL; not less than about 8000 ng/mL; not less than about 8500ng/mL; not less than about 9000 ng/mL; not less than about 9500 ng/mL;not less than about 10000 ng/mL; not less than about 11000 ng/mL; notless than about 12000 ng/mL; not less than about 13000 ng/mL; not lessthan about 14000ng/mL; not less than about 15000 ng/mL; not less thanabout 16000 ng/mL; not less than about 17000 ng/mL; not less than about18000 ng/mL; not less than about 19000 ng/mL; not less than about 20000ng/mL; not less than about 25000 ng/mL; not less than about 30000 ng/mL;not less than about 35000 ng/mL; not less than about 40000 ng/mL; notless than about 45000 ng/mL; not less than about 50000 ng/mL; not lessthan about 55000 ng/mL; not less than about 60000 ng/mL; not less thanabout 65000ng/mL; not less than about 70000 ng/mL; not less than about750000 ng/mL; not less than about 80000 ng/mL; not less than about 85000ng/mL; not less than about 90000 ng/mL; not less than about 95000 ng/mL;not less than about 100000 ng/mL; or any other C. appropriate fordescribing a pharmacokinetic profile of a chlorotoxin conjugatedescribed herein. The C_(max) is, for example, about 1 ng/mL to about100,000 ng/mL; about 1 ng/mL to about 95,00 ng/mL; about 1 ng/mL toabout 90,000 ng/mL; about 1 ng/mL to about 8500 ng/mL; about 1 ng/mL toabout 80000 ng/mL; about 1 ng/mL to about 7500 ng/mL; about 1 ng/mL toabout 70,000 ng/mL; about 1 ng/mL to about 65 00 ng/mL; about 1 ng/mL toabout 60,000 ng/mL; about 1 ng/mL to about 55000 ng/mL; about 1 ng/mL toabout 50000 ng/mL; about 1 ng/mL to about 40000 ng/mL; about 1 ng/mL toabout 30000 ng/mL; about 1 ng/mL to about 20000 ng/mL; about 1 ng/mL toabout 10000 ng/mL; about 1 ng/mL to about 5000 ng/mL; about 1 ng/mL toabout 1000 ng/mL; about 1 ng/mL to about 500 ng/mL; about 1 ng/mL toabout 500 ng/mL; about 1 ng/mL to about 1000 ng/mL; about 1 ng/mL toabout 5000 ng/mL; about 10000 ng/mL to about 5,000 ng/mL; about 10 ng/mLto about 7,000 ng/mL; about 10 ng/mL to about 10,000 ng/mL; about 10ng/mL to about 10,500 ng/mL; about 10 ng/mL to about 100,000 ng/mL;about 10 ng/mL to about 90000 ng/mL; about 10 ng/mL to about 80000ng/mL; about 10 ng/mL to about 70000 ng/mL; about 10 ng/mL to about60000 ng/mL; about 10 ng/mL to about 50000 ng/mL; about 10 ng/mL toabout 40000 ng/mL; about 10 ng/mL to about 30000 ng/mL; about 10 ng/mLto about 20000 ng/mL; about 10 ng/mL to about 10000 ng/mL; about 10ng/mL to about 5000 ng/mL; about 25000 ng/mL to about 50000 ng/mL; about250 ng/mL to about 10000 ng/mL; about 500 ng/mL to about 50000 ng/mL;about 50 ng/mL to about 10000 ng/mL; about 100 ng/mL to about 50000ng/mL; about 100 ng/mL to about 40000 ng/mL; about 100 ng/mL to about30000 ng/mL; or about 100 ng/mL to about 20000 ng/mL.

The plasma concentration of a chlorotoxin conjugate described hereinincludes by way of example but is not limited to, not less than about 1ng/mL, not less than about 2 ng/mL, not less than about 3 ng/mL, notless than about 4 ng/mL, not less than about 5 ng/mL, not less thanabout 6 ng/mL, not less than about 7 ng/mL, not less than about 8 ng/mL,not less than about 9 ng/mL, not less than about 10 ng/mL, not less thanabout 11 ng/mL, not less than about 12 ng/mL, not less than about 13ng/mL, not less than about 14 ng/mL, not less than about 15 ng/mL, notless than about 16 ng/mL, not less than about 17 ng/mL, not less thanabout 18 ng/mL, not less than about 19 ng/mL, not less than about 20ng/mL, not less than about 21 ng/mL, not less than about 22 ng/mL, notless than about 23 ng/mL, not less than about 24 ng/mL, not less thanabout 25 ng/mL, not less than about 26 ng/mL, not less than about 27ng/mL, not less than about 28 ng/mL, not less than about 29 ng/mL, notless than about 30 ng/mL, not less than about 31 ng/mL, not less thanabout 32 ng/mL, not less than about 33 ng/mL, not less than about 34ng/mL, not less than about 35 ng/mL, not less than about 36 ng/mL, notless than about 37 ng/mL, not less than about 38 ng/mL, not less thanabout 39 ng/mL, not less than about 40 ng/mL, not less than about 41ng/mL, not less than about 42 ng/mL, not less than about 43 ng/mL, notless than about 44 ng/mL, not less than about 45 ng/mL, not less thanabout 46 ng/mL, not less than about 47 ng/mL, not less than about 48ng/mL, not less than about 49 ng/mL, not less than about 50 ng/mL, notless than about 51 ng/mL, not less than about 52 ng/mL, not less thanabout 53 ng/mL, not less than about 54 ng/mL, not less than about 55ng/mL, not less than about 56 ng/mL, not less than about 57 ng/mL, notless than about 58 ng/mL, not less than about 59 ng/mL, not less thanabout 60 ng/mL, not less than about 61 ng/mL, not less than about 62ng/mL, not less than about 63 ng/mL, not less than about 64 ng/mL, notless than about 65 ng/mL, not less than about 66 ng/mL, not less thanabout 67 ng/mL, not less than about 68 ng/mL, not less than about 69ng/mL, not less than about 70 ng/mL, not less than about 71 ng/mL, notless than about 72 ng/mL, not less than about 73 ng/mL, not less thanabout 74 ng/mL, not less than about 75 ng/mL, not less than about 76ng/mL, not less than about 77 ng/mL, not less than about 78 ng/mL, notless than about 79 ng/mL, not less than about 80 ng/mL, not less thanabout 81 ng/mL, not less than about 82 ng/mL, not less than about 83ng/mL, not less than about 84 ng/mL, not less than about 85 ng/mL, notless than about 86 ng/mL, not less than about 87 ng/mL, not less thanabout 88 ng/mL, not less than about 89 ng/mL, not less than about 90ng/mL, not less than about 91 ng/mL, not less than about 92 ng/mL, notless than about 93 ng/mL, not less than about 94 ng/mL, not less thanabout 95 ng/mL, not less than about 96 ng/mL, not less than about 97ng/mL, not less than about 98 ng/mL, not less than about 99 ng/mL, notless than about 100 ng/mL, not less than about 105 ng/mL, not less thanabout 110 ng/mL, not less than about 115 ng/mL, not less than about 120ng/mL, not less than about 125 ng/mL, not less than about 130 ng/mL, notless than about 135 ng/mL, not less than about 140 ng/mL, not less thanabout 145 ng/mL, not less than about 150 ng/mL, not less than about 155ng/mL, not less than about 160 ng/mL, not less than about 165 ng/mL, notless than about 170 ng/mL, not less than about 175 ng/mL, not less thanabout 180 ng/mL, not less than about 185 ng/mL, not less than about 190ng/mL, not less than about 195 ng/mL, not less than about 200 ng/mL, notless than about 205 ng/mL, not less than about 210 ng/mL, not less thanabout 215 ng/mL, not less than about 220 ng/mL, not less than about 225ng/mL, not less than about 230 ng/mL, not less than about 235 ng/mL, notless than about 240 ng/mL, not less than about 245 ng/mL, not less thanabout 250 ng/mL, or any other plasma concentration of a chlorotoxinconjugate described herein.

The plasma concentration includes by way of example but is not limitedto, about 1 ng/mL to about 2ng/mL; about 1 ng/mL to about 5 ng/mL; about5 ng/mL to about 10 ng/mL; about 10 ng/mL to about 25 ng/mL; about 25ng/mL to about 50 ng/mL; about 50 ng/mL to about 75 ng/mL; about 75ng/mL to about 100 ng/mL; about 100 ng/mL to about 150 ng/mL; about 100ng/mL to about 200 ng/mL about 150 ng/mL to about 200 ng/mL; about 200ng/mL to about 250 ng/mL; about 250 ng/mL to about 300 ng/mL; about 300ng/mL to about 350 ng/mL; about 350 ng/mL to about 400 ng/mL; about 400ng/mL to about 450 ng/mL; about 450 ng/mL to about 500 ng/mL; about 500ng/mL to about 600 ng/mL; about 600 ng/mL to about 700 ng/mL; about 700ng/mL to about 800 ng/mL; about 800 ng/mL to about 900 ng/mL; about 900ng/mL to about 1,000 ng/mL; about 1,000 ng/mL to about 1,100 ng/mL;about 1,100 ng/mL to about 1,200 ng/mL; about 1,200 ng/mL to about 1,300ng/mL; about 1,300 ng/mL to about 1,400 ng/mL; about 1,400 ng/mL toabout 1,500 ng/mL; about 1,500 ng/mL to about 1,600 ng/mL; about 1,600ng/mL to about 1,700 ng/mL; about 1,700 ng/mL to about 1,800 ng/mL;about 1,800 ng/mL to about 1,900 ng/mL; about 1,900 ng/mL to about 2,000ng/mL; about 2,000 ng/mL to about 3,000 ng/mL; about 3,000 ng/mL toabout 4,000 ng/mL; about 4,000 ng/mL to about 5,000 ng/mL; about 5,000ng/mL to about 6,000 ng/mL; about 6,000 ng/mL to about 7,000 ng/mL;about 7,000 ng/mL to about 8,000 ng/mL; about 8,000 ng/mL to about 9,000ng/mL; or about 9,000 ng/mL to about 10,000 ng/mL.

The T_(max) of a chlorotoxin conjugate described herein includes by wayof example but is not limited to, not greater than about 0.5 minutes,not greater than about 1 minutes, not greater than about 1.5 minutes,not greater than about 2 minutes, not greater than about 2.5 minutes,not greater than about 3 minutes, not greater than about 3.5 minutes,not greater than about 4 minutes, not greater than about 4.5 minutes,not greater than about 5 minutes, or any other Tmax appropriate fordescribing a pharmacokinetic profile of a chlorotoxin conjugatedescribed herein. The T_(max) further includes by way of example but isnot limited to about 0.1 minutes to about 24 minutes; about 0.1 minutesto about 0.5 minutes; about 0.5 minutes to about 1 minute; about 1minute to about 1.5 minutes; about 1.5 minutes to about 2 minute; about2 minutes to about 2.5 minutes; about 2.5 minutes to about 3 minutes;about 3 minutes to about 3.5 minutes; about 3.5 minutes to about 4minutes; about 4 minutes to about 4.5 minutes; about 4.5 minutes toabout 5 minutes; about 5 minutes to about 5.5 minutes; about 5.5 minutesto about 6 minutes; about 6 minutes to about 6.5 minutes; about 6.5minutes to about 7 minutes; about 7 minutes to about 7.5 minutes; about7.5 minutes to about 8 minutes; about 8 minutes to about 8.5 minutes;about 8.5 minutes to about 9 minutes; about 9 minutes to about 9.5minutes; about 9.5 minutes to about 10 minutes; about 10 minutes toabout 10.5 minutes; about 10.5 minutes to about 11 minutes; about 11minutes to about 11.5 minutes; about 11.5 minutes to about 12 minutes;about 12 minutes to about 12.5 minutes; about 12.5 minutes to about 13minutes; about 13 minutes to about 13.5 minutes; about 13.5 minutes toabout 14 minutes; about 14 minutes to about 14.5 minutes; about 14.5minutes to about 15 minutes; about 15 minutes to about 15.5 minutes;about 15.5 minutes to about 16 minutes; about 16 minutes to about 16.5minutes; about 16.5 minutes to about 17 minutes; about 17 minutes toabout 17.5 minutes; about 17.5 minutes to about 18 minutes; about 18minutes to about 18.5 minutes; about 18.5 minutes to about 19 minutes;about 19 minutes to about 19.5 minutes; about 19.5 minutes to about 20minutes; about 20 minutes to about 20.5 minutes; about 20.5 minutes toabout 21 minutes; about 21 minutes to about 21.5 minutes; about 21.5minutes to about 22 minutes; about 22 minutes to about 22.5 minutes;about 22.5 minutes to about 23 minutes; about 23 minutes to about 23.5minutes; about 23.5 minutes to about 24 minutes; about 24 minutes toabout 25 minutes; about 25 minutes to about 25.5 minutes; about 25.5minutes to about 26 minutes; about 26 minutes to about 26.5 minutes;about 26.5 minutes to about 27 minutes; about 27 minutes to about 28minutes; about 28 minutes to about 28.5 minutes; about 28.5 minutes toabout 29 minutes; about 29 minutes to about 29.5 minutes; about 29.5minutes to about 30 minutes; about 30 minutes to about 31 minutes; about31 minutes to about 31.5 minutes; about 31.5 minutes to about 32minutes; about 32 minutes to about 32.5 minutes; about 32.5 minutes toabout 33 minutes; about 33 minutes to about 34 minutes; about 34 minutesto about 35 minutes; about 35 minutes to about 36 minutes; about 36minutes to about 37 minutes; about 37 minutes to about 38 minutes; about38 minutes to about 39 minutes; about 39 minutes to about 40 minutes;about 40 minutes to about 41 minutes; about 41 minutes to about 42minutes; about 42 minutes to about 43 minutes; about 43 minutes to about44 minutes; about 45 minutes to about 46 minutes; about 46 minutes toabout 47 minutes; about 47 minutes to about 48 minutes; about 48 minutesto about 49 minutes; about 49 minutes to about 50 minutes; about 50minutes to about 51 minutes; about 51 minutes to about 52 minutes; about52 minutes to about 53 minutes; about 53 minutes to about 55 minutes;about 55 minutes to about 56 minutes; about 56 minutes to about 57minutes; about 57 minutes to about 58 minutes; about 58 minutes to about59 minutes; about 59 minutes to about 60 minutes; or any other Tmax of achlorotoxin conjugate described herein of a chlorotoxin conjugatedescribed herein.

The T_(max) of a chlorotoxin conjugate described herein includes by wayof example but is not limited to, not greater than about 0.5 hours, notgreater than about 1 hours, not greater than about 1.5 hours, notgreater than about 2 hours, not greater than about 2.5 hours, notgreater than about 3 hours, not greater than about 3.5 hours, notgreater than about 4 hours, not greater than about 4.5 hours, notgreater than about 5 hours, or any other T_(max) appropriate fordescribing a pharmacokinetic profile of a chlorotoxin conjugatedescribed herein. The T_(max) further includes by way of example but isnot limited to about 0.1 hours to about 24 hours; about 0.1 hours toabout 0.5 hours; about 0.5 hours to about 1 hour; about 1 hour to about1.5 hours; about 1.5 hours to about 2 hour; about 2 hours to about 2.5hours; about 2.5 hours to about 3 hours; about 3 hours to about 3.5hours; about 3.5 hours to about 4 hours; about 4 hours to about 4.5hours; about 4.5 hours to about 5 hours; about 5 hours to about 5.5hours; about 5.5 hours to about 6 hours; about 6 hours to about 6.5hours; about 6.5 hours to about 7 hours; about 7 hours to about 7.5hours; about 7.5 hours to about 8 hours; about 8 hours to about 8.5hours; about 8.5 hours to about 9 hours; about 9 hours to about 9.5hours; about 9.5 hours to about 10 hours; about 10 hours to about 10.5hours; about 10.5 hours to about 11 hours; about 11 hours to about 11.5hours; about 11.5 hours to about 12 hours; about 12 hours to about 12.5hours; about 12.5 hours to about 13 hours; about 13 hours to about 13.5hours; about 13.5 hours to about 14 hours; about 14 hours to about 14.5hours; about 14.5 hours to about 15 hours; about 15 hours to about 15.5hours; about 15.5 hours to about 16 hours; about 16 hours to about 16.5hours; about 16.5 hours to about 17 hours; about 17 hours to about 17.5hours; about 17.5 hours to about 18 hours; about 18 hours to about 18.5hours; about 18.5 hours to about 19 hours; about 19 hours to about 19.5hours; about 19.5 hours to about 20 hours; about 20 hours to about 20.5hours; about 20.5 hours to about 21 hours; about 21 hours to about 21.5hours; about 21.5 hours to about 22 hours; about 22 hours to about 22.5hours; about 22.5 hours to about 23 hours; about 23 hours to about 23.5hours; about 23.5 hours to about 24 hours; about 24 hours to about 25hours; about 25 hours to about 25.5 hours; about 25.5 hours to about 26hours; about 26 hours to about 26.5 hours; about 26.5 hours to about 27hours; about 27 hours to about 28 hours; about 28 hours to about 28.5hours; about 28.5 hours to about 29 hours; about 29 hours to about 29.5hours; about 29.5 hours to about 30 hours; about 30 hours to about 31hours; about 31 hours to about 31.5 hours; about 31.5 hours to about 32hours; about 32 hours to about 32.5 hours; about 32.5 hours to about 33hours; about 33 hours to about 34 hours; about 34 hours to about 35hours; about 35 hours to about 36 hours; about 36 hours to about 37hours; about 37 hours to about 38 hours; about 38 hours to about 39hours; about 39 hours to about 40 hours; about 40 hours to about 41hours; about 41 hours to about 42 hours; about 42 hours to about 43hours; about 43 hours to about 44 hours; about 45 hours to about 46hours; about 46 hours to about 47 hours; about 47 hours to about 48hours; about 48 hours to about 49 hours; about 49 hours to about 50hours; about 50 hours to about 51 hours; about 51 hours to about 52hours; about 52 hours to about 53 hours; about 53 hours to about 55hours; about 55 hours to about 56 hours; about 56 hours to about 57hours; about 57 hours to about 58 hours; about 58 hours to about 59hours; about 59 hours to about 60 hours; about 60 hours to about 61hours; about 61 hours to about 62 hours; about 62 hours to about 63hours; about 63 hours to about 64 hours; about 64 hours to about 66hours; about 66 hours to about 67 hours; about 67 hours to about 68hours; about 68 hours to about 69 hours; about 69 hours to about 70hours; about 70 hours to about 71 hours; about 71 hours to about 72hours; about 72 hours to about 73 hours; about 73 hours to about 74hours; about 774hours to about 75 hours; about 75 hours to about 77hours; about 77 hours to about 78 hours; about 78 hours to about 79hours; about79 hours to about 80 hours; about 80 hours to about 81hours; about 81 hours to about 82 hours; about 82 hours to about 83hours; about 83 hours to about 84 hours; about 84 hours to about 85hours; about 85 hours to about 87 hours; about 87 hours to about 88hours; about 88 hours to about 89 hours; about 89 hours to about 90hours; about 90 hours to about 91 hours; about 91 hours to about 92hours; about 92 hours to about 93 hours; about 93 hours to about 94hours; about 94 hours to about 95 hours; about 95 hours to about 97hours; about 97 hours to about 99 hours; about 99 hours to about 100hours; or any other T_(max) of a chlorotoxin conjugate described hereinof a chlorotoxin conjugate described herein.

In some aspects, the chlorotoxin conjugates distribute into the subjecttissues. For example, distribution into the tissues is often rapidcompared to the elimination phase. In some aspects, the chlorotoxinconjugates are eliminated from the subject tissues. For example,elimination from the subject tissues is often slow compared to thedistribution phase. Often the kidney is important in the clearance andelimination of the chlorotoxin conjugates, often contributing to theelimination phase.

The pharmacokinetics parameters are any parameters suitable fordescribing the plasma profiles of chlorotoxin conjugates describedherein and are often associated with a curve. As described elsewhereherein, dose is either scaled or fixed, said scaled dose useful forscaling the dose from one subject to another wherein the subjects arethe same species, different species, same sex or different sex. Thephases of the curve are often representative of data obtained from atleast one subject, sometimes more than one subject, and the phases ofthe curve and/or data of the curve is often scaled in a manner similarto the manner in which doses are scaled.

In some aspects, the curve is plotted on a graph, often a graph with anx-axis and a y-axis referred to for example as an x-y plot, a scatterplot or the like. Each axis of the graph has units, the y-axis oftenhaving units of time, for example in hours, and x-axis often havingunits of concentration, for example as ng/mL, of a chlorotoxin conjugatedescribed herein present in a subject sample as described herein and arerepresentative of a single measurement, a mean, an average, or any othersuitable mathematical calculation performed on a set of data. When asuitable mathematical calculation is performed, a statistic is alsocalculated, for example, a standard error, standard error of the mean,standard deviation, standard deviation of the mean, or any othersuitable statistic useful for the described disclosure.

In some aspects, the curve has phases, for example, distribution phase,metabolism phase and elimination phase. In some aspects, thedistribution phase begins at time of about 0 hours and extends until atime of about 0.01 hours, about 0.02 hours, about 0.03 hours, about 0.04hours, about 0.05 hours, about 0.06 hours, about 0.07 hours, about 0.08hours, about 0.09 hours, about 0.11 hours, about 0.12 hours, about 0.13hours, about 0.14 hours, about 0.15 hours, about 0.16 hours, about 0.17hours, about 0.18 hours, about 0.19 hours, about 0.20 hours, 0.21 hours,about 0.22 hours, about 0.23 hours, about 0.24 hours, about 0.25 hours,about 0.26 hours, about 0.27 hours, about 0.28 hours, about 0.29 hours,about 0.30 hours, about 0.31 hours, about 0.32 hours, about 0.33 hours,about 0.34 hours, about 0.35 hours, about 0.36 hours, about 0.37 hours,about 0.38 hours, about 0.39 hours, about 0.40 hours, about 0.41 hours,about 0.42 hours, about 0.43 hours, about 0.44 hours, about 0.45 hours,about 0.46 hours, about 0.47 hours, about 0.48 hours, about 0.49 hours,about 0.50 hours, about 0.51 hours, about 0.52 hours, about 0.53 hours,about 0.54 hours, about 0.55 hours, about 0.56 hours, about 0.57 hours,about 0.58 hours, about 0.59 hours, about 0.60 hours, about 0.61 hours,about 0.62 hours, about 0.63 hours, about 0.64 hours, about 0.65 hours,about 0.66 hours, about 0.67 hours, about 0.68 hours, about 0.69 hours,about 0.70 hours, about 0.71 hours, about 0.72 hours, about 0.73 hours,about 0.74 hours, about 0.75 hours, about 0.76 hours, about 0.77 hours,about 0.78 hours, about 0.79 hours, about 0.80 hours, about 0.81 hours,about 0.82 hours, about 0.83 hours, about 0.84 hours, about 0.85 hours,about 0.86 hours, about 0.87 hours, about 0.88 hours, about 0.89 hours,about 0.90 hours, about 0.91 hours, about 0.92 hours, about 0.93 hours,about 0.94 hours, about 0.95 hours, about 0.96 hours, about 0.97 hours,about 0.98 hours, about 0.99 hours, about 1.00 hours, about 1.01 hours,about 1.02 hours, about 1.03 hours, about 1.04 hours, about 1.05 hours,about 1.06 hours, about 1.07 hours, about 1.08 hours, about 1.09 hours,about 1.11 hours, about 1.12 hours, about 1.13 hours, about 1.14 hours,about 1.15 hours, about 1.16 hours, about 1.17 hours, about 1.18 hours,about 1.19 hours, about 1.20 hours, 1.21 hours, about 1.22 hours, about1.23 hours, about 1.24 hours, about 1.25 hours, about 1.26 hours, about1.27 hours, about 1.28 hours, about 1.29 hours, about 1.30 hours, about1.31 hours, about 1.32 hours, about 1.33 hours, about 1.34 hours, about1.35 hours, about 1.36 hours, about 1.37 hours, about 1.38 hours, about1.39 hours, about 1.40 hours, about 1.41 hours, about 1.42 hours, about1.43 hours, about 1.44 hours, about 1.45 hours, about 1.46 hours, about1.47 hours, about 1.48 hours, about 1.49 hours, about 1.50 hours, about1.51 hours, about 1.52 hours, about 1.53 hours, about 1.54 hours, about1.55 hours, about 1.56 hours, about 1.57 hours, about 1.58 hours, about1.59 hours, about 1.60 hours, about 1.61 hours, about 1.62 hours, about1.63 hours, about 1.64 hours, about 1.65 hours, about 1.66 hours, about1.67 hours, about 1.68 hours, about 1.69 hours, about 1.70 hours, about1.71 hours, about 1.72 hours, about 1.73 hours, about 1.74 hours, about1.75 hours, about 1.76 hours, about 1.77 hours, about 1.78 hours, about1.79 hours, about 1.80 hours, about 1.81 hours, about 1.82 hours, about1.83 hours, about 1.84 hours, about 1.85 hours, about 1.86 hours, about1.87 hours, about 1.88 hours, about 1.89 hours, about 1.90 hours, about1.91 hours, about 1.92 hours, about 1.93 hours, about 1.94 hours, about1.95 hours, about 1.96 hours, about 1.97 hours, about 1.98 hours, about1.99 hours, about 2.00 hours, about 2.20 hours, about 2.40 hours, about2.60 hours, about 2.80 hours, about 3.00 hours, about 4.20 hours, about4.40 hours, about 4.60 hours, about 4.80 hours, about 5.00 hours, about5.20 hours, about 5.40 hours, about 5.60 hours, about 5.80 hours, about6.00 hours, about 6.20 hours, about 6.40 hours, about 6.60 hours, about6.80 hours, about 7.00 hours, about 7.20 hours, about 7.40 hours, about7.60 hours, about 7.80 hours, about 8.00 hours, about 8.20 hours, about8.40 hours, about 8.60 hours, about 8.80 hours, about 9.00 hours, about9.20 hours, about 9.40 hours, about 9.60 hours, about 9.80 hours, about10.00 hours or more than about 10.00 hours.

In some aspects, the metabolism phase begins at time of about 0.5 hoursand extends until a time of about about 0.50 hours, about 0.51 hours,about 0.52 hours, about 0.53 hours, about 0.54 hours, about 0.55 hours,about 0.56 hours, about 0.57 hours, about 0.58 hours, about 0.59 hours,about 0.60 hours, about 0.61 hours, about 0.62 hours, about 0.63 hours,about 0.64 hours, about 0.65 hours, about 0.66 hours, about 0.67 hours,about 0.68 hours, about 0.69 hours, about 0.70 hours, about 0.71 hours,about 0.72 hours, about 0.73 hours, about 0.74 hours, about 0.75 hours,about 0.76 hours, about 0.77 hours, about 0.78 hours, about 0.79 hours,about 0.80 hours, about 0.81 hours, about 0.82 hours, about 0.83 hours,about 0.84 hours, about 0.85 hours, about 0.86 hours, about 0.87 hours,about 0.88 hours, about 0.89 hours, about 0.90 hours, about 0.91 hours,about 0.92 hours, about 0.93 hours, about 0.94 hours, about 0.95 hours,about 0.96 hours, about 0.97 hours, about 0.98 hours, about 0.99 hours,about 1.00 hours, about 1.01 hours, about 1.02 hours, about 1.03 hours,about 1.04 hours, about 1.05 hours, about 1.06 hours, about 1.07 hours,about 1.08 hours, about 1.09 hours, about 1.11 hours, about 1.12 hours,about 1.13 hours, about 1.14 hours, about 1.15 hours, about 1.16 hours,about 1.17 hours, about 1.18 hours, about 1.19 hours, about 1.20 hours,1.21 hours, about 1.22 hours, about 1.23 hours, about 1.24 hours, about1.25 hours, about 1.26 hours, about 1.27 hours, about 1.28 hours, about1.29 hours, about 1.30 hours, about 1.31 hours, about 1.32 hours, about1.33 hours, about 1.34 hours, about 1.35 hours, about 1.36 hours, about1.37 hours, about 1.38 hours, about 1.39 hours, about 1.40 hours, about1.41 hours, about 1.42 hours, about 1.43 hours, about 1.44 hours, about1.45 hours, about 1.46 hours, about 1.47 hours, about 1.48 hours, about1.49 hours, about 1.50 hours, about 1.51 hours, about 1.52 hours, about1.53 hours, about 1.54 hours, about 1.55 hours, about 1.56 hours, about1.57 hours, about 1.58 hours, about 1.59 hours, about 1.60 hours, about1.61 hours, about 1.62 hours, about 1.63 hours, about 1.64 hours, about1.65 hours, about 1.66 hours, about 1.67 hours, about 1.68 hours, about1.69 hours, about 1.70 hours, about 1.71 hours, about 1.72 hours, about1.73 hours, about 1.74 hours, about 1.75 hours, about 1.76 hours, about1.77 hours, about 1.78 hours, about 1.79 hours, about 1.80 hours, about1.81 hours, about 1.82 hours, about 1.83 hours, about 1.84 hours, about1.85 hours, about 1.86 hours, about 1.87 hours, about 1.88 hours, about1.89 hours, about 1.90 hours, about 1.91 hours, about 1.92 hours, about1.93 hours, about 1.94 hours, about 1.95 hours, about 1.96 hours, about1.97 hours, about 1.98 hours, about 1.99 hours, about 2.00 hours, about2.20 hours, about 2.40 hours, about 2.60 hours, about 2.80 hours, about3.00 hours, about 4.20 hours, about 4.40 hours, about 4.60 hours, about4.80 hours, about 5.00 hours, about 5.20 hours, about 5.40 hours, about5.60 hours, about 5.80 hours, about 6.00 hours, about 6.20 hours, about6.40 hours, about 6.60 hours, about 6.80 hours, about 7.00 hours, about7.20 hours, about 7.40 hours, about 7.60 hours, about 7.80 hours, about8.00 hours, about 8.20 hours, about 8.40 hours, about 8.60 hours, about8.80 hours, about 9.00 hours, about 9.20 hours, about 9.40 hours, about9.60 hours, about 9.80 hours, about 10.00 hours, about 10.20 hours,about 10.40 hours, about 10.60 hours, about 10.80 hours, about 12.00hours, about 12.20 hours, about 12.40 hours, about 12.60 hours, about12.80 hours, about 14.00 hours, about 14.20 hours, about 14.40 hours,about 14.60 hours, about 14.80 hours, about 16.00 hours, about 16.20hours, about 16.40 hours, about 16.60 hours, about 16.80 hours, about18.00 hours, about 18.20 hours, about 18.40 hours, about 18.60 hours,about 18.80 hours, about 20.00 hours, about 20.20 hours, about 20.40hours, about 20.60 hours, about 20.80 hours, about 22.00 hours, about22.20 hours, about 22.40 hours, about 22.60 hours, about 22.80 hours,about 24.00 hours, about 24.20 hours, about 24.40 hours, about 24.60hours, about 24.80 hours, about 26.00 hours, about 26.20 hours, about26.40 hours, about 26.60 hours, about 26.80 hours, about 28.00 hours,about 28.20 hours, about 28.40 hours, about 28.60 hours, about 28.80hours, about 30 hours or more than about 30.00 hours.

In some aspects, the elimination phase begins at time of about 2 hoursand extends until a time of about 2.00 hours, about 2.20 hours, about2.40 hours, about 2.60 hours, about 2.80 hours, about 3.00 hours, about4.20 hours, about 4.40 hours, about 4.60 hours, about 4.80 hours, about5.00 hours, about 5.20 hours, about 5.40 hours, about 5.60 hours, about5.80 hours, about 6.00 hours, about 6.20 hours, about 6.40 hours, about6.60 hours, about 6.80 hours, about 7.00 hours, about 7.20 hours, about7.40 hours, about 7.60 hours, about 7.80 hours, about 8.00 hours, about8.20 hours, about 8.40 hours, about 8.60 hours, about 8.80 hours, about9.00 hours, about 9.20 hours, about 9.40 hours, about 9.60 hours, about9.80 hours, about 10.00 hours, about 10.20 hours, about 10.40 hours,about 10.60 hours, about 10.80 hours, about 12.00 hours, about 12.20hours, about 12.40 hours, about 12.60 hours, about 12.80 hours, about14.00 hours, about 14.20 hours, about 14.40 hours, about 14.60 hours,about 14.80 hours, about 16.00 hours, about 16.20 hours, about 16.40hours, about 16.60 hours, about 16.80 hours, about 18.00 hours, about18.20 hours, about 18.40 hours, about 18.60 hours, about 18.80 hours,about 20.00 hours, about 20.20 hours, about 20.40 hours, about 20.60hours, about 20.80 hours, about 22.00 hours, about 22.20 hours, about22.40 hours, about 22.60 hours, about 22.80 hours, about 24.00 hours,about 24.20 hours, about 24.40 hours, about 24.60 hours, about 24.80hours, about 26.00 hours, about 26.20 hours, about 26.40 hours, about26.60 hours, about 26.80 hours, about 28.00 hours, about 28.20 hours,about 28.40 hours, about 28.60 hours, about 28.80 hours, about 30.00hours, about 30.20 hours, about 30.40 hours, about 30.60 hours, about30.80 hours, about 32.00 hours, about 32.20 hours, about 32.40 hours,about 32.60 hours, about 32.80 hours, about 34.00 hours, about 34.20hours, about 34.40 hours, about 34.60 hours, about 34.80 hours, about36.00 hours, about 36.20 hours, about 36.40 hours, about 36.60 hours,about 36.80 hours, about 38.00 hours, about 38.20 hours, about 38.40hours, about 38.60 hours, about 38.80 hours, about 40.00 hours, about40.20 hours, about 40.40 hours, about 40.60 hours, about 40.80 hours,about 42.00 hours, about 42.20 hours, about 42.40 hours, about 42.60hours, about 42.80 hours, about 44.00 hours, about 44.20 hours, about44.40 hours, about 44.60 hours, about 44.80 hours, about 46.00 hours,about 46.20 hours, about 46.40 hours, about 46.60 hours, about 46.80hours, about 48.00 hours, about 48.20 hours, about 48.40 hours, about48.60 hours, about 48.80 hours, about 50.00 hours, about 50.20 hours,about 50.40 hours, about 50.60 hours, about 50.80 hours, about 52.00hours, about 52.20 hours, about 52.40 hours, about 52.60 hours, about52.80 hours, about 54.00 hours, about 54.20 hours, about 54.40 hours,about 54.60 hours, about 54.80 hours, about 56.00 hours, about 56.20hours, about 56.40 hours, about 56.60 hours, about 56.80 hours, about58.00 hours, about 58.20 hours, about 58.40 hours, about 58.60 hours,about 58.80 hours, about 60.00 hours, about 60.20 hours, about 60.40hours, about 60.60 hours, about 60.80 hours, about 62.00 hours, about62.20 hours, about 62.40 hours, about 62.60 hours, about 62.80 hours,about 64.00 hours, about 64.20 hours, about 64.40 hours, about 64.60hours, about 64.80 hours, about 66.00 hours, about 66.20 hours, about66.40 hours, about 66.60 hours, about 66.80 hours, about 68.00 hours,about 68.20 hours, about 68.40 hours, about 68.60 hours, about 68.80hours, about 70.00 hours, about 70.20 hours, about 70.40 hours, about70.60 hours, about 70.80 hours, about 72.00 hours, about 72.20 hours,about 72.40 hours, about 72.60 hours, about 72.80 hours, about 74.00hours, about 74.20 hours, about 74.40 hours, about 74.60 hours, about74.80 hours, about 76.00 hours, about 76.20 hours, about 76.40 hours,about 76.60 hours, about 76.80 hours, about 78.00 hours, about 78.20hours, about 78.40 hours, about 78.60 hours, about 78.80 hours, about80.00 hours, about 80.20 hours, about 80.40 hours, about 80.60 hours,about 80.80 hours, about 82.00 hours, about 82.20 hours, about 82.40hours, about 82.60 hours, about 82.80 hours, about 84.00 hours, about84.20 hours, about 84.40 hours, about 84.60 hours, about 84.80 hours,about 86.00 hours, about 86.20 hours, about 86.40 hours, about 86.60hours, about 86.80 hours, about 88.00 hours, about 88.20 hours, about88.40 hours, about 88.60 hours, about 88.80 hours, about 90.00 hours orabout more than 90.00 hours.

In some aspects, a single fixed bolus dose intravenous chlorotoxinconjugate often results in mean serum concentrations measurable up toabout 12 hours post-dose, about 24 hours post-dose, up to about 36 hourspost-dose, up to about 48 hours post-dose or more than about 48 hourspost-dose. Often, for subjects such as rats, C_(max) and C₀ parametersincrease in about a dose-proportional manner. In some aspects, theAUC_(0-t) parameter, for subjects such as rats, is aboutdose-proportional at less than about a 1 mg dose levels, and increasesin a greater than dose-proportional manner at greater than about 1 mgdose levels. Often there is no effect of gender on any PK parameters forsubjects such as rats. In some aspects, PK parameters are predictive inrats of a human subject.

In some aspects, a single fixed bolus dose intravenous chlorotoxinconjugate often results in mean serum concentrations measurable up toabout 12 hours post-dose, about 24 hours post-dose, up to about 36 hourspost-dose, up to about 48 hours post-dose or more than about 48 hourspost-dose. Often, for subjects such as rats, C_(max) and C₀ parametersincrease in about a dose-proportional manner. In some aspects, theAUC_(0-t) parameter, for subjects such as monkeys, is greater thandose-proportional manner at greater than about 1 mg dose levels forexample such that chlorotoxin conjugates exhibit reduced clearance athigher doses in monkeys. Often there is an effect of gender on PKparameters for subjects such as monkeys, for example, the C₀ and AUC areabout 5 to about 30% higher in females relative to males.

As used herein, two pharmacokinetic profiles are “about equivalent” ifthey are defined by at least one parameter that is about equivalentbetween the two profiles. Non-limiting examples of such parametersinclude the area under plasma concentration over time curve (AUC) andthe maximal plasma concentration reached following administration of adose (C_(max)).

In some aspects two pharmacokinetic parameters are about equivalent ifthe lower value is greater than 70%, greater than 75%, greater than 80%,greater than 85%, greater than 90%, greater than 95%, greater than 96%,greater than 97%, greater than 98%, or greater than 99% of the highervalue.

The pharmacokinetic profiles of two dosage regimens are compared bydetermining the average pharmacokinetic profile in a population ofsubjects receiving the first dosage regimen, determining the averagepharmacokinetic profile in a population of subjects receiving the seconddosage regimen, and then comparing those two population dosage regimens.In some aspects, a population of subjects is one subject. In otheraspects, a population of subjects is more than one subject, for example,two subjects, three subjects, four subjects, five subjects, sixsubjects, seven subjects, eight subjects, nine subjects, ten subjects,11 subjects, 12 subjects, 13 subjects, 14 subjects, 15 subjects, 20subjects, 25 subjects, 30 subjects, 35 subjects, 40 subjects, 45subjects, 50 subjects, or more than 50 subjects.

In various aspects, the present disclosure provides a method ofadministering a composition to a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof; and producing in the human subject an average maximumcompound blood plasma concentration (average C_(max))of at least from110 ng/mL to 240 ng/mL per each 1 mg dosage of the compoundadministered.

In various aspects, the present disclosure provides a method ofdetecting a cancer cell in a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof conjugated to a detectable label; producing in thehuman subject an average maximum compound blood plasma concentration(average C_(max))of at least from 110 ng/mL to 240 ng/mL per each 1 mgdosage of the compound administered; and detecting the presence orabsence of the detectable label in the human subject, wherein thepresence of the detectable label indicates the presence of the cancercell.

In various aspects, the present disclosure provides a method ofdiagnosing cancer in a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof conjugated to a detectable label; producing in thehuman subject an average maximum compound blood plasma concentration(average C_(max))of at least from 110 ng/mL to240 ng/mL per each 1 mgdosage of the compound administered; and detecting the presence orabsence of the detectable label in the human subject, wherein thepresence of the detectable label indicates a diagnosis of cancer.

In various aspects, the present disclosure provides a method of treatingcancer in a human subject, the method comprising: intravenouslyadministering to the human subject a dose of from 1 mg to 30 mg of acompound comprising a polypeptide having at least 85% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereofconjugated to a therapeutic agent; producing in the human subject anaverage maximum compound blood plasma concentration (average C_(max))ofat least from 110 ng/mL to 240 ng/mL per each 1 mg dosage of thecompound administered; and reducing or improving a symptom or conditionassociated with cancer in the human subject. In some aspects, the humansubject is in need thereof In some aspects, the methods compriseadministering a therapeutically effective dose of the compound to thehuman subject.

In various aspects, the present disclosure provides a method ofadministering a composition to a human subject, the method comprising:administering to the human subject a dose of from 1 mg to 30 mg of acompound comprising a polypeptide having at least 85% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof andproducing in the human subject pharmacokinetic profile of FIG. 27 .

In various aspects, the present disclosure provides a method ofadministering a composition to a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of any suitable compound of the present disclosure; and producingin the human subject an average maximum compound blood plasmaconcentration (average C_(max))of at least from 110 ng/mL to240 ng/mLper each 1 mg dosage of the compound administered.

In some aspects, the average time (average T_(max)) at which the averageC_(max) is reached is at 5±4 minutes following administration of thecompound. In some aspects, the average time (average T₇₅) at which theaverage compound blood plasma concentration reaches 75% of the averageC_(max) (average C75) is reached is at 8±5 minutes followingadministration of the compound. In some aspects, the average time(average T₅₀) at which the average compound blood plasma concentrationreaches 50% of the average C_(max) (average C₅₀) is reached is at 20±8minutes following administration of the compound. In some aspects, theaverage time (average T₂₅) at which the average compound blood plasmaconcentration reaches 25% of the average C_(max) (average C₂₅) isreached is at 30±12 minutes following administration of the compound.

In some aspects, the methods further comprise producing in the humansubject an average chlorotoxin polypeptide plasma area under the curve(average AUC) of from 50 hr*ng/mL to 120 hr*ng/mL per each 1 mg dosageof chlorotoxin polypeptide administered.

In some aspects, the methods further comprise producing in the humansubject an average chlorotoxin polypeptide plasma area under the curve(average AUC) of from 60 hr*ng/mL to 110 hr*ng/mL per each 1 mg dosageof chlorotoxin polypeptide administered.

In some aspects, 75% of the average AUC occurs within 40±15 minutesafter administering the compound. In some aspects, 50% of the averageAUC occurs within 21±8 minutes after administering the compound. In someaspects, 25% of the average AUC occurs within 9±5 minutes afteradministering the compound.

In some aspects, the compound comprises any suitable compound of thepresent disclosure.

In various aspects, the present disclosure provides a method fordetecting a cancer cell in a subject, the method comprising:administering any suitable compound of the present disclosure; anddetecting the presence or absence of the compound in the subject,wherein the presence of the compound indicates the presence of a cancercell.

In some aspects, the method further comprises administering the compoundas a part of a composition.

In some aspects, the cancer is selected from glioma, astrocytoma,medulloblastoma, choroids plexus carcinoma, ependymoma, brain tumor,neuroblastoma, adenocarcinoma, basal cell carcinoma, squamous cellcarcinoma, head and neck cancer, lung cancer, breast cancer, intestinalcancer, pancreatic cancer, liver cancer, kidney cancer, sarcoma,osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinoma, melanoma,ovarian cancer, cervical cancer, lymphoma, thyroid cancer, anal cancer,colo-rectal cancer, endometrial cancer, germ cell tumor, laryngealcancer, multiple myeloma, prostate cancer, retinoblastoma, gastriccancer, testicular cancer, or Wilm's tumor. In some aspects, the canceris selected from glioma, medulloblastoma, sarcoma, breast cancer, lungcancer, prostate cancer, or intestinal cancer. In some aspects, thecancer cell expresses a site to which native chlorotoxin binds.

In some aspects, the method comprises detecting the compound byfluorescence imaging.

In some aspects, the method further comprises differentiating a focus ofa cancer that expresses a site to which native chlorotoxin binds fromnon-neoplastic tissue.

In some aspects, the method further comprises surgically removing fromthe subject a cancer cell that is detected.

In some aspects, the method further comprises determining the locationof a cancer cell in the subject before surgically removing the cancercell from the subject, during surgical removal of the cancer cell fromthe subject, after removing the cancer cell from the subject, or acombination thereof.

In some aspects, the compound binds to the cancer cell. In some aspects,the subject is a human subject. In some aspects, the detection isperformed in vivo or ex vivo.

In various aspects, the present disclosure provides a method ofadministering any suitable compound of the present disclosure to asubject, the method comprising administering a therapeutically effectiveamount of the compound to the subject.

In some aspects, the subject is in need thereof.

In some aspects, a therapeutically effective amount is a dosagesufficient for the detection of a cancer cell in the subject. In someaspects, the dosage is from 0.1 mg to 100 mg. In some aspects, dosage isfrom 1 mg to 30 mg. In some aspects, the dosage is from 3 mg to 30 mg.

In various aspects, the present disclosure provides a method of treatinga subject in need thereof, the method comprising administering to thesubject any suitable compound of the present disclosure furthercomprising a therapeutic agent in an amount sufficient to treat cancerin the subject. In certain aspects, the therapeutic agent is a cytotoxicagent.

In some aspects, the cancer is selected from glioma, astrocytoma,medulloblastoma, choroids plexus carcinoma, ependymoma, brain tumor,neuroblastoma, head and neck cancer, lung cancer, breast cancer,intestinal cancer, pancreatic cancer, liver cancer, kidney cancer,sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinoma,melanoma, ovarian cancer, cervical cancer, lymphoma, thyroid cancer,anal cancer, colo-rectal cancer, endometrial cancer, germ cell tumor,laryngeal cancer, multiple myeloma, prostate cancer, retinoblastoma,gastric cancer, testicular cancer, or Wilm's tumor. In some aspects, thecancer cell is selected from glioma, medulloblastoma, sarcoma, prostatecancer, or intestinal cancer. In certain aspects, the cancer cellexpresses a site to which native chlorotoxin binds. In further aspects,the binding is selective.

In some aspects, the compound is administered parenterally. In otheraspects, the compound is administered intravenously. In still otheraspects, the compound is administered subcutaneously.

Methods for Analysis to Generate Pharmacokinetic Profiles

In some aspects, samples are analyzed to obtain parameters useful todetermine a pharmacokinetic profile. Often the samples are diluted, forexample, using a buffer or pharmaceutically acceptable carrier asdefined herein.

Pharmacokinetic standard curves are often generated using a chlorotoxinconjugate, serum and a pharmaceutical carrier as described herein. Theproportion of each chlorotoxin conjugate, concentrated source of sample(for example serum, urine, etc.) and pharmaceutical carrier oftendiffers, for example, the concentration of compound of the presentdisclosure is often between about 10 μg/mL and about 4 ng/mL. Often thestandard curve is used to calculate the concentration of the compound inthe sample.

In some aspects, pharmacokinetic parameters, or pharmacokinetic data areanalyzed using standard pharmacokinetic data analysis methods, includingconcentration of chlorotoxin conjugates versus time. For example, asoftware program, such as Phoenix WinNonlin 6.3 is used to analyzepharmacokinetics data. In some aspects, the pharmacokinetic dataanalysis uses standard noncompartmental methods of intravenous bolus,intravenous infusion, or extravascular input as appropriate. In otheraspects, the pharmacokinetic data analysis uses nonstandardnoncompartmental methods of intravenous bolus, intravenous infusion, orextravascular input as appropriate. Often, the data are analyzed by themean serum concentration versus time. The data are also analyzed byindividual subject followed by group summary statistics.

Pharmacokinetic profiles of the compositions described herein are oftenobtained using at least one, sometimes more than one bioanalyticalmethod. In some aspects, bioanalytical methods include the addition ofchemicals to a sample containing a composition of which thepharmacokinetic profile is desired. Addition of the chemical to thesample often comprises performing a chemical technique to measure theconcentration of a composition or a metabolite thereof in a sample or,sometimes, in a biological matrix. For example, microscalethermophoresis, mass spectrometry often including liquid chromatorgraphand a triple quadropole mass spectrometer, tandem mass spectrometry,high sensitivity mass spectrometry for microdosing studeies and the likeare often performed.

The disclosure further describes methods of administering compound andcompositions of the present disclsoure to a subject, often methodsinclude intravenous administration of a chlorotoxin conjugatecomposition to a subject. In some aspects, the method of administering achlorotoxin polypeptide to a subject comprises intravenouslyadministering a dose of from 0.8 to 25 mg of the chlorotoxin polypeptideto the subject, wherein the chlorotoxin polypeptide has at least 85%sequence identity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR; producingin the subject an average chlorotoxin polypeptide plasma area under thecurve (average AUC) of from 50 to 120 per each 1 mg dosage ofchlorotoxin polypeptide administered; and producing in the subject anaverage maximum chlorotoxin peptide blood plasma concentration (averageC_(max))of at least from 110 to 240 per each 1 mg dosage of chlorotoxinpeptide administered. In some aspects, the chlorotoxin polypeptide isconjugated to a fluorescent agent.

The disclosure further describes methods of treating and/or detectingcancer with chlorotoxin conjugate compositions following administrationto a subject or contacting tumor tissue isolated from a subject, oftenthe methods include intravenous administration of a chlorotoxinconjugate composition to a subject, in vivo contact of a tumor tissue orex vivo contact of a tumor tissue from a subject. In some aspects, themethod treating and/or detecting cancer with chlorotoxin conjugatecompositions following administration to a subject or contacting tumortissue isolated from a subject, often the methods include intravenousadministration of a chlorotoxin conjugate composition to a subject, invivo contact of a tumor tissue or ex vivo contact of a tumor tissue froma subject comprises intravenously administering a dose of from 0.8 to 25mg of the chlorotoxin polypeptide to the subject, wherein thechlorotoxin polypeptide has at least 85% sequence identity withMCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR; producing in the subject anaverage chlorotoxin polypeptide plasma area under the curve (averageAUC) of from 50 to 120 per each 1 mg dosage of chlorotoxin polypeptideadministered; and producing in the subject an average maximumchlorotoxin peptide blood plasma concentration (average C_(max))of atleast from 110 to 240 per each 1 mg dosage of chlorotoxin peptideadministered. In some aspects, the chlorotoxin polypeptide is conjugatedto a fluorescent agent.

The disclosure further describes compositions of chlorotoxin conjugates,often the chlorotoxin conjugate composition comprises a physiologicallyeffective amount of a chlorotoxin conjugate, wherein the chlorotoxinconjugate comprises a chlorotoxin polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR conjugatedto a fluorescent dye, for example, as provided in the presentdislcosure, or a derivative thereof, wherein intravenous administrationof the composition to a subject produces in the subject: an averagechlorotoxin polypeptide plasma area under the curve (average AUC) offrom 50 to 120 per each 1 mg dosage of chlorotoxin polypeptideadministered; and an average maximum chlorotoxin peptide blood plasmaconcentration (average C_(max))of at least from 110 to 240 per each 1 mgdosage of chlorotoxin peptide administered. In some aspects, thechlorotoxin polypeptide is conjugated to a fluorescent agent.

Activity of Chlorotoxin Conjugates

The present disclosure provides, but is not limited to, methods forintraoperative imaging and resection of tumors with chlorotoxinconjugates detectable by fluorescence imaging that allows forintraoperative visualization of cancerous tissues, compositions thatinclude the chlorotoxin conjugate, and methods for using the chlorotoxinconjugate. The chlorotoxin is a targeting agent that directs theconjugate to a tissue of interest. In one aspect, the chlorotoxinconjugate of the disclosure includes one or more labeling agents. In afurther aspect, the labeling agent comprises a fluorescent moiety (e.g.,red or near infrared emitting fluorescent moieties) covalently coupledto the chlorotoxin. In another aspect, the labeling agent comprises aradionuclide.

The chlorotoxin conjugates described herein are often used for detectionand treatment of, for example imaging, resection of, diagnosis of andtreatment of tumors. In some aspects, tumors amenable to detection witha chlorotoxin conjugate of the present disclosure include, but are notlimited to: adenocarcinoma, fibrosarcoma, hemangiosarcoma, mastocytoma,squamous cell carcinoma, chondrosarcoma, adenosquamous carcinoma,hemangiopericytoma, follicular carcinoma, meningioma, mucosal squamouscell cancer, glioma, sarcomas, such as soft-tissue sarcomas or the like.Soft-tissue sarcomas amenable to detection with a chlorotoxin conjugateof the present disclosure include, but are not limited to: fat tissuetumors, liposarcomas, muscle tissue tumors including smooth musclesarcomas and leiomyosarcomas, skeletal muscle sarcomas,rhabdomyosarcomas, peripheral nerve tumors, fibrous tissue tumors,myxofibrosarcomas, fibromatosis, joint tissue tumors, tumors of bloodvessels and lymph vessels, angiosarcomas, tumors of peripheral nervessuch as malignant peripheral nerve sheath tumors, malignant schwannomas,neurofibrosarcomas, fibrosarcomas, synovial sarcomas, malignant fibroushistiocytoma (MFH) hemangiosarcomas, lymphangiosarcomas,gastrointestinal stromal tumors, alveolar soft part sarcoma,dermatofibrosarcoma protuberans (DFSP), desmoplastic small round celltumour, epithelioid sarcoma, extra skeletal myxoid chondrosarcoma, andgiant cell fibroblastoma (GCF).

The chlorotoxin conjugates described herein can be used for detectionand treatment of tumors present in any organ and in any anatomicallocation, including but not limited to, breast, lung, brain, colon,rectum, prostate, head, neck, stomach, anus, and/or vaginal tissues, forexample. Tumors of any grade or stage known to one of skill in the art,including low-grade tumors, are often detected by the chlorotoxinconjugates described herein. In some aspects, tumor detection includesimaging, resection, diagnostics and treatment.

In certain aspects, the present compounds are capable of passing acrossthe blood brain barrier. Passing across the blood brain barrier isadvantageous when detecting or treating a cancer cell in the brain, suchas for example, a glioma cell or a brain tumor.

In some aspects, the dose of chlorotoxin conjugate is administered suchthat a threshold amount of chlorotoxin conjugate is achieved in thesubject. For example, the threshold amount often depends upon thepatient's age, weight, height, sex, general medical condition andprevious medical history. For another example, the threshold amount doesnot depend upon the patient's age, weight, height, sex, general medicalcondition and previous medical history.

Other dosage forms is devised by those skilled in the art, as shown, forexample, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5th Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions are often supplied as akit comprising a container that comprises a chlorotoxin conjugate.Chlorotoxin conjugates are often provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit oftenincludes a dry-powder disperser, liquid aerosol generator, or nebulizerfor administration of a therapeutic conjugate. Such a kit furthercomprises written information on indications and usage of thepharmaceutical composition.

Methods of Tumor Prevention, Detection and Treatment

Subjects include, but are not limited to humans, non-human primates,monkeys, cows, dogs, rabbits, pigs, guinea pigs, rats, mice andzebrafish.

A sample includes any sample isolated from a subject, for example butnot limited to, blood, serum, plasma, circulating cells, urine, saliva,and/or tissue removed from the body such as in a biopsy. Samples areoften prepared using methods known to those of ordinary skill in theart, for example, blood samples are collected and incubated at roomtemperature for about 0.5 hours up to 2 hours prior to centrifugation,serum removal and storage at at least about 20° C., but more often −70°C.

Additional tests are often performed using samples from subjects,including complete blood counts, serum chemistry profiles andurinalysis.

The present disclosure provides methods for treating a disease orcondition treatable by administering chlorotoxin. In one embodiment, themethod includes administering an effective amount of a modifiedchlorotoxin peptide of the invention to a subject in need thereof.

The term “effective amount,” as used herein, refers to a sufficientamount of an agent or a compound being administered which will relieveto some extent one or more of the symptoms of the disease or conditionbeing treated. The result can be reduction and/or alleviation of thesigns, symptoms, or causes of a disease, or any other desired alterationof a biological system. Compositions containing such agents or compoundscan be administered for prophylactic, enhancing, and/or therapeutictreatments. An appropriate “effective” amount in any individual case maybe determined using techniques, such as a dose escalation study.

In one embodiment, the invention provides a method for treating a cancerthat expresses chlorotoxin binding sites in a patient, comprisingadministering to a patient in need thereof an effective amount of achlorotoxin variant of the invention.

In one embodiment, the invention provides a method for treating a cancerthat expresses chlorotoxin binding sites, comprising administering to apatient in need thereof an effective amount of a pharmaceuticalcomposition comprising a chlorotoxin variant of the invention and apharmaceutically acceptable carrier.

In one embodiment, the invention provides a method for treating a tumorexpressing chlorotoxin binding sites, comprising administering to apatient in need thereof an effective amount of a chlorotoxin variant ofthe invention.

In one embodiment, the invention provides a method for inhibitinginvasive activity of cells that express chlorotoxin binding sites,comprising administering an effective amount of a chlorotoxin variant tocells that express chlorotoxin binding sites.

The methods of treatment of the invention are applicable to human andanimal subjects in need of such treatment.

Virtually every type of malignant cancer expressing chlorotoxin bindingsites can be treated by the chlorotoxin variants and conjugates of theinvention. These malignant cancers include gliomas, astrocytomas,medulloblastomas, choroid plexus carcinomas, ependymomas, meningioma,glioblastoma, ganglioma, pheochromocytoma, and metastatic brain tumors,other brain tumors, neuroblastoma, head and neck cancer, non-small celllung cancer, small cell lung cancer, breast cancer, intestinal cancer,pancreatic cancer, colon cancer, liver cancer, kidney cancer, skincancer, sarcomas (over 30 types), osteosarcoma, rhabdomyosarcoma,Ewing's sarcoma, carcinomas, melanomas, ovarian cancer, cervical cancer,lymphoma, thyroid cancer, anal cancer, colo-rectal cancer, endometrialcancer, germ cell tumors, laryngeal cancer, multiple myeloma, prostatecancer, retinoblastoma, gastric cancer, testicular cancer, and Wilm'stumor.

In certain aspects, the chlorotoxin conjugate is administered to anindividual having or suspected of having a tumor, such that theconjugate binds specifically to the tumor. Such methods are useful inreducing the likelihood that the individual will develop a tumor, thatone or more tumors in the individual will increase in size, that one ormore tumors in the individual will metastasize, and/or that the cancerwill progress by some other measure. As used herein, the term“metastasis” refers to the spread of tumor cells from one organ ortissue to another location, and also refers to tumor tissue that formsin a new location as a result of metastasis.

In some aspects, the chlorotoxin conjugate is useful for the treatmentand/or diagnosis of neuroectodermal tumors such as gliomas,medulloblastomas, neuroblastomas, pheochromocytomas, melanomas,peripheral primitive neuroectodermal tumors, small cell carcinoma of thelung, Ewing's sarcoma, and metastatic tumors in the brain. In someaspects, the chlorotoxin conjugate is useful for the treatment and/ordiagnosis of brain tumors, including but not limited to, glioma,including glioblastoma multiforme, anaplastic astrocytomas, low gradegliomas, pliocytic astrocytomas, oligodendrogliomas, gangliomas,meningiomas, and ependymomas.

In other aspects, the compounds of the present disclosure are used todetect and/or treat soft-tissue sarcomas. Soft-tissue sarcomas are agroup of malignant tumors that form in fat, muscles, nerves, joints, andblood vessels. In 2012, it was estimated that approximately 11,280Americans would be diagnosed with soft-tissue sarcomas and approximately3,900 would be expected to die from soft-tissue sarcomas. Soft tissueand bone sarcoma incidence rates have increased slightly over the past30 years; however, soft-tissue sarcoma is more deadly, possibly becausethe lack of specific symptoms at early disease stages may lead to delaysin diagnosis.

Moreover, certain inherited disorders and past treatment with radiationtherapy can increase the risk of soft-tissue sarcoma. No modifiable riskfactors for sarcoma have been identified. Standard treatments forsoft-tissue sarcoma include surgery, chemotherapy, and radiationtherapy.

Since symptoms of soft-tissue sarcomas often do not appear until thedisease is advanced, only about 50% of soft-tissue sarcomas are found inthe early stages, before they have spread.

The present invention provides such methods of detection, imaging,visualization, analysis and treatment for these and other uses thatshould be apparent to those skilled in the art from the teachingsherein.

The present invention is based in part upon the identification by theinventors that soft-tissue sarcomas have a high level of uptake of theconjugate compared to other tumors or normal tissues and areparticularly well-suited for detection by means of administering achlorotoxin conjugated to a labeling agent for detection, visualization,imaging, or analysis. Such visualization can be during or related tosurgical (intraoperative) resection or during or related to initialidentification of the sarcoma or during or related to monitoring of thesarcoma relevant to treatment.

Real-time intraoperative visualization of solid tumors enables morecomplete resection while sparing surrounding normal tissue. Improvementin intraoperative tumor visualization would be of benefit for anyresectable solid tumor, as it would enable surgeons to better determinethe extent of local invasion as well as the presence of metastaticspread in nearby lymph nodes and fatty tissue. This kind of informationwould be helpful in making surgical decisions, for example, decisionsregarding which patients would respond well to limb-sparing approachesin the treatment of sarcomas.

For brain tumors this is of paramount importance, since removal ofadditional tissue can unnecessarily increase cognitive and functionalimpairment, yet being too conservative in the amount of resection mayleave tumor tissue behind.

Surgeons who specialize in human breast cancer surgery have indicatedthat precise margins are less important in this indication, and thesurgical approach is generally a wide excision with 0.2-1 cm margins onall sides. It is difficult for surgeons to obtain wide margins usingonly white light and preoperative imaging information. In 20-50% ofbreast cancer surgeries, failure to obtain clean margins leads to secondsurgeries.

The present invention shows that soft-tissue sarcomas are particularlyidentifiable when bound by a chlorotoxin conjugate, which can beeffectively used to detect these sarcomas, especially during or relatedto surgery and intraoperative resection. For example, the chlorotoxinconjugate can be used alone or on combination with other detectionagents, to detect, image, visualize, or analyze the tumor in advance of,during, or following anti-tumor treatments, which can include surgeryand surgical resection, chemotherapy, radiation therapy, andimmunotherapy. In addition, the chlorotoxin conjugate can be used aloneor with other detection agents for follow-up monitoring post treatmentas well as for general monitoring for full-body screening.

Low-grade tumors generally tend to be slow growing, slower to spread,and often have better prognosis than higher-grade tumors, making themmore curable with surgical resection than high-grade tumors, which mayneed more systemic treatment. The inventors show that use of achlorotoxin conjugated to a labeling agent can be particularly effectivein detection, imaging, visualization, or analysis of low-grade tumors,such as meningiomas, allowing their complete resection before theymetastasize or spread.

Intraoperative resection of tumor types may vary depending on theanatomic location and type of tumor. For example, when a tumor islocated in brain tissue, the surgeon is likely to require perfect ornear perfect specificity between a tumor imaging or detection agent andthe tumor tissue so that only diseased tissue is resected. On the otherhand, when the tumor is located in a tissue where wider margins aregenerally resected, such as breast or mammary cancer or colon cancer, orin cancers where the tumor is likely to spread locally, such as squamouscell carcinoma, it would be advantageous for a surgeon to be able to usea tumor imaging or detection agent to identify peritumoral tissue thatis likely to become tumor tissue.

Soft-tissue sarcomas can develop from soft tissues like fat, muscle,nerves, fibrous tissues, blood vessels, or deep skin tissues. They canbe found in any part of the body. Most of them develop in the arms orlegs. They can also be found in the trunk, head and neck area, internalorgans, and the area in back of the abdominal cavity (known as theretroperitoneum).

Soft-tissue sarcomas that may be amenable to detection with achlorotoxin conjugate of the present invention include, but are notlimited to: fat tissue tumors, muscle tissue tumors, skeletal musclesarcomas, rhabdomyosarcomas, peripheral nerve tumors, fibrous tissuetumors, myxofibrosarcomas, fibromatosis, joint tissue tumors, tumors ofblood vessels and lymph vessels, angiosarcomas, gastrointestinal stromaltumors, alveolar soft part sarcoma, dermatofibrosarcoma protuberans(DFSP), desmoplastic small round cell tumour, epithelioid sarcoma, extraskeletal myxoid chondrosarcoma, and giant cell fibroblastoma (GCF).

Sarcomas that start in the body's fat cells are called liposarcomas.They can grow anywhere in the body and most commonly affect people aged50-65 years. Some grow very slowly, taking many years to develop,whereas others grow more quickly.

Muscle tissue sarcomas include smooth muscle sarcomas and skeletalmuscle sarcomas. Smooth muscle forms the walls of internal organs suchas the stomach, intestine, womb (uterus), and blood vessels. The musclecauses these organs to contract, which happens without our control.Smooth muscle is also called involuntary muscle. Sarcomas that developin smooth muscle are called leiomyosarcomas. They are one of the morecommon types of sarcoma and can occur anywhere in the body, especiallyin the back of the abdominal area (retroperitoneum). Leiomyosarcomas areless often found in the deep, soft tissues of the legs or arms. Theytend to occur in adults, particularly in the elderly. Skeletal musclesare the active muscles in our arms and legs or other parts of the bodythat we control. They are voluntary muscles and sometimes calledstriated muscles because the cells look stripy when examined under amicroscope.

Sarcomas that grow in the voluntary muscles of the body are calledrhabdomyosarcomas. They are found mostly in the head and neck, but alsoin organs such as the bladder, vagina and the arms or legs.Rhabdomyosarcomas are more commonly diagnosed in children than inadults.

Peripheral nerve tumors can be found in the peripheral nervous system,which consists of all the nerves that run throughout the body. Sarcomasof the peripheral nerves develop in the cells that cover the nerves.They're known as malignant peripheral nerve sheath tumors (MPNST) andcan occur anywhere in the body. There are different types of MPNSTs,including malignant schwannomas and neurofibrosarcomas. They mostcommonly occur in people who have a rare genetic disorder calledneurofibromatosis (von Recklinghausen's disease).

Fibrous tissue tumors occur in tissues that join muscles to bones. Thistissue is made up of cells called fibrocytes. A sarcoma of the fibroustissue is called a fibrosarcoma. They are most commonly found on thearms, legs or trunk, but can occur deeper in the body. They can occur atany age but are more commonly seen in people aged 20-60 years. Mostpeople first notice them as a painless, firm lump.

Soft-tissue sarcomas that develop very close to the body's joints areknown as synovial sarcomas. They commonly develop near, but not inside,joints such as the knee or elbow, but they can occur in any part of thebody. They usually appear as hard lumps and are more common in childrenand young adults.

Blood and lymph vessel tumors include sarcomas that start from the cellsthat make up the walls of blood or lymph vessels and are calledangiosarcomas. Haemangiosarcomas develop from blood vessels andlymphangiosarcomas develop from the lymph vessels.

Angiosarcomas are sarcomas that sometimes occur in a part of the bodythat has been treated with radiotherapy many years before.

Gastrointestinal stromal tumors (GIST) are soft-tissue sarcomas thatdevelop in nerve cells in the walls of the digestive system.

The inventors have also identified that low-grade tumors can be detectedwith a chlorotoxin conjugated to a labeling agent. This is particularlyuseful since low-grade tumors have a better prognosis if they can befully resected.

In addition, the invention is based in part on the identification by theinventors of an optimal dose for tumor imaging in dogs of at least about0.8 mg/m². One skilled in the art will recognize that dosage for thechlorotoxin conjugate will be determined based on the amount ofconjugate administered and the amount of time after administration afterwhich the imaging is performed. In some aspects the optimal dose fortumor imaging is in the range of about 0.85 mg/m² to about 1.2 mg/m², orin the range of about 0.9 to about 1.1 mg/m². At doses up to 0.9 mg/m²,signal in gross tumor samples increases as a function of dose. At dosesabove 0.9 mg/m², the correlation between signal and dose is lost,suggesting that in this model system the signal in tumor is maximalabove this dose. However, it is recognized that in other cases moreconjugate can be administered with acceptable imaging. Thus, in someaspects, the amount of chlorotoxin conjugate that can be administeredcan be in the range of about 1.3 mg/m² to about 2.5 mg/m², or in therange of about 2.6 mg/m² to about 3.5 mg/m², or in the range of about3.6 mg/m² to about 4.5 mg/m², or in the range of about 4.6 mg/m² toabout 5.5 mg/m², or upwards of 5.5 mg/m².

Imaging Methods

In a further aspect of the invention, methods of using the chlorotoxinconjugates are provided. In one embodiment, the invention provides amethod for imaging a tissue imagable by chlorotoxin. In the method, atissue imagable by chlorotoxin is contacted with a chlorotoxinconjugate. In one embodiment, the imaging method is a fluorescenceimaging method. Representative methods for making and using fluorescentchlorotoxin conjugates are described in U.S. Patent ApplicationPublication No. 20080279780 A1, Fluorescent Chlorotoxin Conjugate andMethod for Intra-Operative Visualization of Cancer, and in U.S. PatentApplication Publication No. 20130195760, Chlorotoxin Variants,Conjugates, And Methods For Their Use, both of which are expresslyincorporated herein by reference in their entirety.

In many cases, chlorotoxin conjugates can be administered to human andanimal subjects, such as with a pharmaceutically acceptable carrier. Insome aspects, the composition includes a pharmacologically effectiveamount of a modified chlorotoxin conjugate. An effective amount can beroutinely determined by established procedures. An effective amount isan amount sufficient to occupy chlorotoxin binding sites in cancercells, but low enough to minimize non-specific binding to non-neoplastictissues. An effective amount optimizes signal-to-noise ratio forintra-operative imaging.

The disclosure provides methods for detecting a tissue using thechlorotoxin conjugates. The chlorotoxin conjugates of the inventiontarget and are bound by chlorotoxin binding sites. It will beappreciated that chlorotoxin binding sites may take two forms: sitesthat bind chlorotoxin and sites that bind the chlorotoxin conjugates ofthe invention. It will be appreciated that chlorotoxin binding sites maybe distinct from chlorotoxin conjugate binding sites.

In some aspects, a method for differentiating foci of cancers thatexpress chlorotoxin binding sites from non-neoplastic tissue isprovided. The method includes contacting a tissue of interest with achlorotoxin conjugate having affinity and specificity for cells thatexpress chlorotoxin binding sites, wherein the chlorotoxin conjugatecomprises one or more red or near infrared emitting fluorescent moietiescovalently coupled to a chlorotoxin, and measuring the level of bindingof the chlorotoxin conjugate, wherein an elevated level of binding,relative to normal tissue, is indicative that the tissue is neoplastic.

In some aspects, a method for detecting cancers that express chlorotoxinbinding sites is provided. The method includes the steps of contacting atissue of interest with a chlorotoxin conjugate having affinity andspecificity for cells that express chlorotoxin binding sites, whereinthe chlorotoxin conjugate comprises one or more red or near infraredemitting fluorescent moieties covalently coupled to a chlorotoxin, andmeasuring the level of binding of the chlorotoxin conjugate, wherein anelevated level of binding, relative to normal tissue, is indicative thatthe tissue is neoplastic.

In some aspects, a method for determining the location of cancer cellsthat express chlorotoxin binding sites in a patient intra-operatively isprovided. The method includes the steps of administering apharmaceutical composition to a patient, wherein the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier and anamount of a chlorotoxin conjugate sufficient to image cancer cells thatexpress chlorotoxin binding sites in vivo, wherein the chlorotoxinconjugate comprises one or more red or near infrared emittingfluorescent moieties covalently coupled to a chlorotoxin, measuring thelevel of binding of the chlorotoxin conjugate by fluorescence imaging todetermine the location of cancer cells that express chlorotoxin bindingsites, wherein an elevated level of binding, relative to normal tissue,is indicative of the presence of cancer cells that express chlorotoxinbinding sites; and surgically removing from the patient at least somecells that express chlorotoxin binding sites located by fluorescenceimaging.

Imaging methods for detection of cancer foci disclosed herein areapplicable to mouse and other animal models of cancer as well as toveterinary practice.

The present invention provides methods for intraoperative imaging andresection of tumors with a chlorotoxin conjugates detectable byfluorescence imaging that allows for intraoperative visualization ofcancerous tissues, compositions that include the chlorotoxin conjugate,and methods for using the chlorotoxin conjugate. The chlorotoxin is atargeting agent that directs the conjugate to a tissue of interest. Inone embodiment, the chlorotoxin conjugate of the invention includes oneor more labeling agents. In a further embodiment, the labeling agentcomprises a fluorescent moiety (e.g., red or near infrared emittingfluorescent moieties) covalently coupled to the chlorotoxin. In anotherembodiment, the labeling agent comprises a radionuclide.

As used herein, the term “red or near infrared emitting fluorescentmoiety” refers to a fluorescent moiety having a fluorescence emissionmaximum greater than about 600 nm.

In certain embodiments of the chlorotoxin conjugate, the fluorescentmoieties are derived from fluorescent compounds characterized byemission wavelength maxima greater than about 600 nm to avoidautofluorescence, emission that travels through millimeters to onecentimeter of tissue/blood/fluids, emission that is not absorbed byhemoglobin, other blood components, or proteins in human or animaltissue. In some aspects, the emission wavelength maximum is greater than600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm,greater than 800 nm, greater than 850 nm, greater than 900 nm, orgreater than 950 nm.

The fluorescent moiety is covalently coupled to the chlorotoxin to allowfor the visualization of the conjugate by fluorescence imaging. Thefluorescent moiety is derived from a fluorescent compound. Suitablefluorescent compounds are those that can be covalently coupled to achlorotoxin without substantially adversely affecting the targeting andbinding function of the chlorotoxin conjugate. Similarly, suitablefluorescent compounds retain their fluorescent properties afterconjugation to the chlorotoxin.

Generally, the dosage of administered chlorotoxin conjugates may varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of chlorotoxinconjugated to a chemotherapeutic, an anti-cancer agent, or ananti-cancer drug that is effective to achieve inhibition, shrinkage,killing, minimization, or prevention of metastasis. In many cases, it isdesirable to provide the recipient with a dosage of a chlorotoxinconjugate that is in the range of from about 3 mg to about 6 mg,although a lower or higher dosage also may be administered ascircumstances dictate.

Administration of a chlorotoxin conjugate to a subject can be topical,inhalant, intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringconjugates by injection, the administration may be by continuousinfusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Ilium, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising achlorotoxin conjugate can be prepared and inhaled with the aid ofdry-powder dispersers, liquid aerosol generators, or nebulizers (e.g.,Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. DrugDeliv. Rev. 35:235 (1999)). This approach is illustrated by the AERXdiabetes management system, which is a hand-held electronic inhaler thatdelivers aerosolized insulin into the lungs. Transdermal delivery usingelectroporation provides another means to administer a chlorotoxinconjugate.

A pharmaceutical composition comprising a chlorotoxin conjugate can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the conjugate is combined with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

A pharmaceutical composition comprising a chlorotoxin conjugate can befurnished in liquid form, in an aerosol, or in solid form. Liquid forms,are illustrated by injectable solutions, aerosols, droplets, topologicalsolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Othersolid forms include creams, pastes, other topological applications, andthe like.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5.sup.th Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19.sup.th Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a chlorotoxin conjugate.Therapeutic conjugates can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic conjugate. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition.

Various references, including patent applications, patents, andscientific publications, are cited herein, the disclosures of each ofwhich is incorporated herein by reference in its entirety.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

All features discussed in connection with any aspect or embodimentherein can be readily adapted for use in other aspects and embodimentsherein. The use of different terms or reference numerals for similarfeatures in different embodiments does not necessarily imply differencesother than those expressly set forth. Accordingly, the present inventionis intended to be described solely by reference to the appended claims,and not limited to the embodiments disclosed herein.

Unless otherwise specified, the presently described methods andprocesses can be performed in any order. For example, a methoddescribing steps (a), (b), and (c) can be performed with step (a) first,followed by step (b), and then step (c). Or, the method can be performedin a different order such as, for example, with step (b) first followedby step (c) and then step (a). Furthermore, those steps can be performedsimultaneously or separately unless otherwise specified withparticularity.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure provided herein. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe disclosure provided herein.

All features discussed in connection with an aspect or embodiment hereincan be readily adapted for use in other aspects and embodiments herein.The use of different terms or reference numerals for similar features indifferent embodiments does not necessarily imply differences other thanthose expressly set forth. Accordingly, the present invention isintended to be described solely by reference to the appended claims, andnot limited to the embodiments disclosed herein.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Numbered Embodiments

The following embodiments recite non-limiting permutations ofcombinations of feature disclosed herein. Other permutations ofcombinations of features are alos contemplated. In particular, each ofthese numbered embodiments is contemplated as depending from or relatingto every previous or subsequent numbered embodiment. 1. A peptide activeagent conjugate, comprising: a) a peptide, wherein the peptide comprisesa sequence that has at least 70% sequence identity with any one of SEQID NO: 508—SEQ ID NO: 758 and upon administration to a subject thepeptide homes, targets, migrates to, accumulates in, binds to, isretained by, or is directed to a cartilage of the subject, and an activeagent selected from an active agent class selected from TABLE 53 orTABLE 55; b) a peptide, wherein the peptide comprises a sequence thathas at least 70% sequence identity with any one of SEQ ID NO: 508—SEQ IDNO: 758 and upon administration to a subject the peptide homes, targets,migrates to, accumulates in, binds to, is retained by, or is directed toa kidney of the subject, and an active agent selected from an activeagent class selected from TABLE 54 or TABLE 55; c) a peptide, whereinthe peptide comprises a sequence that has at least 70% sequence identitywith any one of SEQ ID NO: 508—SEQ ID NO: 758 and upon administration toa subject the peptide homes, targets, migrates to, accumulates in, bindsto, is retained by, or is directed to a cartilage or kidney of thesubject, and an active agent selected from TABLE 53, TABLE 54, or TABLE55; d) a peptide, wherein the peptide comprises a sequence that has atleast 70% sequence identity with any one of SEQ ID NO: 798—SEQ ID NO:1048 and upon administration to a subject the peptide homes, targets,migrates to, accumulates in, binds to, is retained by, or is directed toa cartilage of the subject, and an active agent selected from TABLE 53or TABLE 55; e) a peptide, wherein the peptide comprises a sequence thathas at least 70% sequence identity with any one of SEQ ID NO: 798—SEQ IDNO: 1048 and upon administration to a subject the peptide homes,targets, migrates to, accumulates in, binds to, is retained by, or isdirected to a kidney of the subject, and an active agent selected froman active agent class selected from TABLE 54 or TABLE 55; 0 a peptide,wherein the peptide comprises a sequence that has at least 70% sequenceidentity with any one of SEQ ID NO: 798—SEQ ID NO: 1048 and uponadministration to a subject the peptide homes, targets, migrates to,accumulates in, binds to, is retained by, or is directed to a cartilageor kidney of the subject, and an active agent selected from an activeagent class selected from TABLE 53, TABLE 54, or TABLE 55; g) a peptide,wherein the peptide comprises a sequence that has at least 70% sequenceidentity with any one of SEQ ID NO: 744—SEQ ID NO: 758 and uponadministration to a subject the peptide homes, targets, migrates to,accumulates in, binds to, is retained by, or is directed to a cartilageof the subject, and an active agent selected from TABLE 53, TABLE 55, orTABLE 56; h) a peptide, wherein the peptide comprises a sequence thathas at least 70% sequence identity with any one of SEQ ID NO: 744—SEQ IDNO: 758 and upon administration to a subject the peptide homes, targets,migrates to, accumulates in, binds to, is retained by, or is directed toa kidney of the subject, and an active agent selected from TABLE 54,TABLE 55, or TABLE 56; i) a peptide, wherein the peptide comprises asequence that has at least 70% sequence identity with any one of SEQ IDNO: 744—SEQ ID NO: 758 and upon administration to a subject the peptidehomes, targets, migrates to, accumulates in, binds to, is retained by,or is directed to a cartilage or a kidney of the subject, and an activeagent selected from TABLE 53, TABLE 54, TABLE 55, or TABLE 56; j) apeptide, wherein the peptide comprises a sequence that has at least 70%sequence identity with any one of SEQ ID NO: 1034—SEQ ID NO: 1048 andupon administration to a subject the peptide homes, targets, migratesto, accumulates in, binds to, is retained by, or is directed to acartilage of the subject, and an active agent selected from TABLE 53,TABLE 55, or TABLE 56; k) a peptide, wherein the peptide comprises asequence that has at least 70% sequence identity with any one of SEQ IDNO: 1034- SEQ ID NO: 1048 and upon administration to a subject thepeptide homes, targets, migrates to, accumulates in, binds to, isretained by, or is directed to a kidney of the subject, and an activeagent selected from TABLE 54, TABLE 55, or TABLE 56; or 1) a peptide,wherein the peptide comprises a sequence that has at least 70% sequenceidentity with any one of SEQ ID NO: 1034—SEQ ID NO: 1048 and uponadministration to a subject the peptide homes, targets, migrates to,accumulates in, binds to, is retained by, or is directed to a cartilageor a kidney of the subject, and an active agent selected from TABLE 53,TABLE 54, TABLE 55, or TABLE 56. 2. The peptide active agent conjugateof embodiment 1, wherein the peptide comprises: a) a sequence that hasat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99% or 100% sequence identity with any one of SEQ IDNO: 508—SEQ ID NO: 758 or a fragment thereof; b) a sequence that has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99% or 100% sequence identity with any one of SEQ IDNO: 744—SEQ ID NO: 758 or a fragment thereof; c) a sequence that has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99% or 100% sequence identity with any one of SEQ IDNO: 798—SEQ ID NO: 1048 or a fragment thereof; or d) a sequence that hasat least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 99% or 100% sequence identity with any one of SEQ IDNO: 1034—SEQ ID NO: 1048 or a fragment thereof 3. The peptide activeagent conjugate of any one of embodiments 1-2, wherein the peptidecomprises: a) a sequence of any one of SEQ ID NO: 508—SEQ ID NO: 758 ora fragment thereof; b) a sequence of any one of SEQ ID NO: 744—SEQ IDNO: 758 or a fragment thereof; c) a sequence of any one of SEQ ID NO:798—SEQ ID NO: 1048 or a fragment thereof; or d) a sequence of any oneof SEQ ID NO: 1034—SEQ ID NO: 1048 or a fragment thereof. 4. A peptidecomprising a sequence that has at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or100% sequence identity with any one of SEQ ID NO: 744—SEQ ID NO: 758 orat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 99%, or 100% sequence identity withany one of SEQ ID NO: 1034—SEQ ID NO: 1048. 5. The peptide active agentconjugate of any one of embodiments 1-3 or the peptide of embodiment 4,wherein the peptide comprises: a) a sequence of any one of SEQ ID NO:485- SEQ ID NO: 507 or a fragment thereof; b) a sequence of any one ofSEQ ID NO: 759—SEQ ID NO: 781 or a fragment thereof; c) a sequence ofany one of SEQ ID NO: 505—SEQ ID NO: 507 or a fragment thereof; or d) asequence of any one of SEQ ID NO: 779—SEQ ID NO: 781 or a fragmentthereof 6. The peptide active agent conjugate of any one of embodiments1-3 or 5, or the peptide of any one of embodiments 4-5, wherein thepeptide is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least, 97%, atleast 98%, or at least 99% identical to any one of SEQ ID NO: 978—SEQ IDNO: 1024 or at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% identical to any one of SEQ ID NO: 688—SEQ IDNO: 734. 7. The peptide active agent conjugate of any one of embodiments1-3 or 5-6 or the peptide of any one of embodiments 4-6, wherein thepeptide is at least 70%, at least 80%, at least 90%, at least 95%, atleast 97%, or 100% identical to: a) SEQ ID NO: 595; b) SEQ ID NO: 885;c) SEQ ID NO: 508; d) SEQ ID NO: 798; e) SEQ ID NO: 511; f) SEQ ID NO:801; g) SEQ ID NO: 669; h) SEQ ID NO: 959; i) SEQ ID NO: 514; j) SEQ IDNO: 804; k) SEQ ID NO: 592; 1) SEQ ID NO: 882; m) SEQ ID NO: 520; n) SEQID NO: 810; o) SEQ ID NO: 683; p) SEQ ID NO: 962; q) SEQ ID NO: 509; r)SEQ ID NO: 799; s) SEQ ID NO: 590; t) SEQ ID NO: 880; u) SEQ ID NO: 510;v) SEQ ID NO: 800; w) SEQ ID NO: 671; x) SEQ ID NO: 961; y) SEQ ID NO:591; or z) SEQ ID NO: 881. 8. The peptide active agent conjugate of anyone of embodiments 1-3 or 5-7 or the peptide of any one of embodiments4-7, wherein the peptide is at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, at least 99%, or 100% identical to: a) SEQ IDNO: 1034; b) SEQ ID NO: 1035; c) SEQ ID NO: 1036; d) SEQ ID NO: 1037; e)SEQ ID NO: 1038; f) SEQ ID NO: 1039; g) SEQ ID NO: 1040; h) SEQ ID NO:1041; i) SEQ ID NO: 1042; j) SEQ ID NO: 1043; k) SEQ ID NO: 1044; 1) SEQID NO: 1045; m) SEQ ID NO: 1046; n) SEQ ID NO: 1047; o) SEQ ID NO: 1048;p) SEQ ID NO: 744; q) SEQ ID NO: 745; r) SEQ ID NO: 746; s) SEQ ID NO:747; t) SEQ ID NO: 748; u) SEQ ID NO: 749; v) SEQ ID NO: 750; w) SEQ IDNO: 751; x) SEQ ID NO: 752; y) SEQ ID NO: 753; z) SEQ ID NO: 754; aa)SEQ ID NO: 755; bb) SEQ ID NO: 756; cc) SEQ ID NO: 757; or dd) SEQ IDNO: 758. 9. The peptide active agent conjugate of any one of embodiments1-3 or 5-8 or the peptide of any one of embodiments 4-8, wherein thepeptide homes, targets, migrates to, accumulates in, binds to, isretained by, or is directed to cartilage, to kidney, or to cartilage andkidney. 10. The peptide active agent conjugate of any one of embodiments1-3 or 5-9 or the peptide of any one of embodiments 4-9, wherein thepeptide homes, targets, migrates to, accumulates in, binds to, isretained by, or is directed to proximal tubules of the kidney. 11. Thepeptide active agent conjugate of any one of embodiments 1-3 or 5-10 orthe peptide of any one of embodiments 4-10, wherein the peptide iscovalently conjugated to the active agent. 12. The peptide active agentconjugate of any one of embodiments 1-3 or 5-11, wherein the peptideactive agent conjugate homes, targets, migrates to, accumulates in,binds to, is retained by, or is directed to a cartilage or a kidney ofthe subject. 13. The peptide active agent conjugate of any one ofembodiments 1-3 or 5-12 or the peptide of any one of embodiments 4-12,wherein the peptide comprises 4 or more cysteine residues. 14. Thepeptide active agent conjugate of any one of embodiments 1-3 or 5-13 orthe peptide of any one of embodiments 4-13, wherein the peptidecomprises three or more disulfide bridges formed between cysteineresidues, wherein one of the disulfide bridges passes through a loopformed by two other disulfide bridges. 15. The peptide active agentconjugate of any one of embodiments 1-3 or 5-14 or the peptide of anyone of embodiments 4-14, wherein the peptide comprises a plurality ofdisulfide bridges formed between cysteine residues. 16. The peptideactive agent conjugate of any one of embodiments 1-3 or 5-15 or thepeptide of any one of embodiments 4-15, wherein the peptide comprises adisulfide through a disulfide knot. 17. The peptide active agentconjugate of any one of embodiments 1-3 or 5-16 or the peptide of anyone of embodiments 4-16, wherein at least one amino acid residue of thepeptide is in an L configuration or, wherein at least one amino acidresidue of the peptide is in a D configuration. 18. The peptide activeagent conjugate of any of embodiments 1-3 or 5-17 or the peptide of anyone of embodiments 4-17, wherein the sequence comprises at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, at least 30, at least 31, at least 32, at least 33, atleast 34, at least 35, at least 36, at least 37, at least 38, at least39, at least 40, at least 41, at least 42, at least 43, at least 44, atleast 45, at least 46, at least 47, at least 48, at least 49, at least50, at least 51, at least 52, at least 53, at least 54, at least 55, atleast 56, at least 57, at least 58 residues, at least 59, at least 60,at least 61, at least 62, at least 63, at least 64, at least 65, atleast 66, at least 67, at least 68, at least 69, at least 70, at least71, at least 72, at least 73, at least 74, at least 75, at least 76, atleast 77, at least 78, at least 79, at least 80, or at least 81residues. 19. The peptide active agent conjugate of any of embodiments1-3 or 5-18 or the peptide of any one of embodiments 4-18, wherein anyone or more K residues are replaced by an R residue or wherein any oneor more R residues are replaced by for a K residue. 20. The peptideactive agent conjugate of any of embodiments 1-3 or 5-19 or the peptideof any one of embodiments 4-19, wherein any one or more M residues arereplaced by any one of the I, L, or V residues. 21. The peptide activeagent conjugate of any of embodiments 1-3 or 5-20 or the peptide of anyone of embodiments 4-20, wherein any one or more L residues are replacedby any one of the V, I, or M residues. 22. The peptide active agentconjugate of any of embodiments 1-3 or 5-21 or the peptide of any one ofembodiments 4-21, wherein any one or more I residues are replaced by anyof the M, L, or V residues. 23. The peptide active agent conjugate ofany of embodiments 1-3 or 5-22 or the peptide of any one of embodiments4-22, wherein any one or more V residues are replaced by any of the M,I, or L residues. 24. The peptide active agent conjugate of any ofembodiments 1-3 or 5-23 or the peptide of any one of embodiments 4-23,wherein any one or more G residues are replaced by an A residue orwherein any one or more A residues are replaced by a G residue. 25. Thepeptide active agent conjugate of any of embodiments 1-3 or 5-24 or thepeptide of any one of embodiments 4-24, wherein any one or more Sresidues are replaced by a T residue or wherein any one or more Tresidues are replaced by for an S residue. 26. The peptide active agentconjugate of any of embodiments 1-3 or 5-25 or the peptide of any one ofembodiments 4-25, wherein any one or more Q residues are replaced by anN residue or wherein any one or more N residues are replaced by a Qresidue. 27. The peptide active agent conjugate of any of embodiments1-3 or 5-26 or the peptide of any one of embodiments 4-26, wherein anyone or more D residues are replaced by an E residue or wherein any oneor more E residues are replaced by a D residue. 28. The peptide activeagent conjugate of any of any one of embodiments 1-3 or 5-27 or thepeptide of any one of embodiments 4-27, wherein the peptide has a chargedistribution comprising an acidic region and a basic region. 29. Thepeptide of embodiment 28, wherein the acidic region is a nub. 30. Thepeptide of embodiment 28, wherein the basic region is a patch. 31. Thepeptide active agent conjugate of any one of embodiments 1-3 or 5-30 orthe peptide of any one of embodiments 4-30, wherein the peptidecomprises 5-12 basic residues. 32. The peptide active agent conjugate ofany one of embodiments 1-3 or 5-31 or the peptide of any one ofembodiments 4-31, wherein the peptide comprises 0-5 acidic residues. 33.The peptide active agent conjugate of any of embodiments 1-3 or 5-32 orthe peptide of any one of embodiments 4-32, wherein the peptidecomprises 6 or more basic residues and 2 or fewer acidic residues. 34.The peptide active agent conjugate of any of embodiments 1-3 or 5-33 orthe peptide of any one of embodiments 4-33, wherein the peptidecomprises a 4-19 amino acid residue fragment containing at least 2cysteine residues, and at least 2 positively charged amino acidresidues. 35. The peptide active agent conjugate of any of embodiments1-3 or 5-34 or the peptide of any one of embodiments 4-34, wherein thepeptide comprises a 20-70 amino acid residue fragment containing atleast 2 cysteine residues, no more than 2 basic residues and at least 2positively charged amino acid residues. 36. The peptide active agentconjugate of any of embodiments 1-3 or 5-35 or the peptide of any one ofembodiments 4-35, wherein the peptide comprises at least 3 positivelycharged amino acid residues. 37. The peptide active agent conjugate ofany of embodiments 34-36 or the peptide of any one of embodiments 34-36,wherein the positively charged amino acid residues are selected from K,R, or a combination thereof 38. The peptide active agent conjugate ofany one of embodiments 1-3 or 5-37 or the peptide of any one ofembodiments 4-37, wherein the peptide has a charge greater than 2 atphysiological pH. 39. The peptide active agent conjugate of any one ofembodiments 1-3 or 5-38 or the peptide of any one of embodiments 4-38,wherein the peptide has a charge greater than 3.5 at physiological pH.40. The peptide active agent conjugate of any one of embodiments 1-3 or5-39 or the peptide of any one of embodiments 4-39, wherein the peptidehas a charge greater than 4.5 at physiological pH. 41. The peptideactive agent conjugate of any one of embodiments 1-3 or 5-40 or thepeptide of any one of embodiments 4-40, wherein the peptide has a chargegreater than 5.5 at physiological pH. 42. The peptide active agentconjugate of any one of embodiments 1-3 or 5-41 or the peptide of anyone of embodiments 4-41, wherein the peptide has a charge greater than6.5 at physiological pH. 43. The peptide active agent conjugate of anyone of embodiments 1-3 or 5-42 or the peptide of any one of embodiments4-42, wherein the peptide has a charge greater than 7.5 at physiologicalpH. 44. The peptide active agent conjugate of any one of embodiments 1-3or 5-43 or the peptide of any one of embodiments 4-43, wherein thepeptide has a charge greater than 8.5 at physiological pH. 45. Thepeptide active agent conjugate of any one of embodiments 1-3 or 5-44 orthe peptide of any one of embodiments 4-44, wherein the peptide has acharge greater than 9.5 at physiological pH. 46. The peptide activeagent conjugate of any one of embodiments 1-3 or 5-45 or the peptide ofany one of embodiments 4-45, wherein the peptide is selected from apotassium channel agonist, a potassium channel antagonist, a portion ofa potassium channel, a sodium channel agonist, a sodium channelantagonist, a calcium channel agonist, a calcium channel antagonist, ahadrucalcin, a theraphotoxin, a huwentoxin, a kaliotoxin, a cobatoxin,or a lectin. 47. The peptide active agent conjugate of embodiment 46 orpeptide of embodiment 46, wherein the lectin is SHL-Ib2. 48. The peptideactive agent conjugate of any one of embodiments 1-3 or 5-47 or thepeptide of any one of embodiments 4-47, wherein the peptide is arrangedin a multimeric structure with at least one other peptide. 49. Thepeptide active agent conjugate of any one of embodiments 1-3 or 5-48 orthe peptide of any one of embodiments 4-48, wherein at least one residueof the peptide comprises a chemical modification. 50. The peptide activeagent conjugate of embodiment 49 or the peptide of embodiment 49,wherein the chemical modification is blocking the N-terminus of thepeptide. 51. The peptide active agent conjugate of embodiment 49 or thepeptide of embodiment 49, wherein the chemical modification ismethylation, acetylation, or acylation. 52. The peptide active agentconjugate of embodiment 49 or the peptide of embodiment 49, wherein thechemical modification is: methylation of one or more lysine residues oranalogue thereof; methylation of the N-terminus; or methylation of oneor more lysine residue or analogue thereof and methylation of theN-terminus. 53. The peptide active agent conjugate of any one ofembodiments 1-3 or 5-52 or the peptide of any one of embodiments 4-52,wherein the peptide is linked to an acyl adduct. 54. The peptide activeagent conjugate of any one of embodiments 1-3 or 5-53 or the peptide ofany one of embodiments 4-53, wherein the peptide is linked to an activeagent. 55. The peptide active agent conjugate of embodiment 54, whereinthe active agent is fused with the peptide at an N-terminus or aC-terminus of the peptide. 56. The peptide active agent conjugate ofembodiment 55, wherein the active agent is another peptide. 57. Thepeptide active agent conjugate of embodiment 56, wherein the activeagent is an antibody. 58. The peptide active agent conjugate ofembodiment 56, wherein the active agent is an Fc domain, Fab domain,scFv, or Fv fragment. 59. The peptide active agent conjugate of any oneof embodiments 55-58, wherein the peptide fused with an Fc domaincomprises a contiguous sequence. 60. The peptide active agent conjugateof any one of embodiments 1-3 or 5-59 or the peptide of any one ofembodiments 4-59, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 active agentsare linked to the peptide. 61. The peptide active agent conjugate of anyone of embodiments 1-3 or 5-60, wherein the peptide is linked to theactive agent at an N-terminus, at the epsilon amine of an internallysine residue, at the carboxylic acid of an aspartic acid or glutamicacid residue, or a C-terminus of the peptide by a linker. 62. Thepeptide active agent conjugate of any one of embodiments 1-3 or 5-61,wherein the peptide is linked to the active agent via a cleavablelinker. 63. The peptide active agent conjugate of any one of embodiments1-3 or 5-62 or the peptide of any one of embodiments 4-62 furthercomprising a non-natural amino acid, wherein the non-natural amino acidis an insertion, appendage, or substitution for another amino acid. 64.The peptide active agent conjugate of embodiment 63 or peptide ofembodiment 63, wherein the peptide is linked to the active agent at thenon-natural amino acid by a linker. 65. The peptide active agentconjugate of any one of embodiments 1-3 or 5-64, wherein the linkercomprises an amide bond, an ester bond, a carbamate bond, a carbonatebond, a hydrazone bond, an oxime bond, a disulfide bond, a thioesterbond, a thioether bond, a triazole, a carbon-carbon bond, or acarbon-nitrogen bond. 66. The peptide active agent conjugate ofembodiment 62, wherein the cleavable linker comprises a cleavage sitefor matrix metalloproteinases, thrombin, cathepsins, orbeta-glucuronidase. 67. The peptide active agent conjugate of any one ofembodiments 61-66, wherein the linker is a hydrolytically labile linker.68. The peptide active agent conjugate of any one of embodiments 61-67,wherein the linker is pH sensitive, reducible, glutathione-sensitive, orprotease cleavable. 69. The peptide active agent conjugate of any one ofembodiments 1-3 or 5-68, wherein the peptide is linked to the activeagent via a stable linker. 70. The peptide active agent conjugate of anyone of embodiments 1-3 or 5-69 or the peptide of any one of embodiments4-69, wherein the peptide has an isoelectric point of about 9. 71. Thepeptide active agent conjugate of any one of embodiments 1-3 or 5-70 orpeptide of any one of embodiments 4-70, wherein the peptide is linked toa detectable agent. 72. The peptide active agent conjugate or peptide ofembodiment 71, wherein the detectable agent is fused with the peptide atan N-terminus or a C-terminus of the peptide. 73. The peptide activeagent conjugate or peptide any one of embodiments 71-72, wherein 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 detectable agents are linked to the peptide.74. The peptide active agent conjugate or peptide of any one ofembodiments 71-73, wherein the peptide is linked to the detectable agentvia a cleavable linker. 75. The peptide active agent conjugate orpeptide of any one of embodiments 71-74, wherein the peptide is linkedto the detectable agent at an N-terminus, at the epsilon amine of aninternal lysine residue, or a C-terminus of the peptide by a linker. 76.The peptide active agent conjugate or peptide of any one of embodiments71-75, further comprising a non-natural amino acid, wherein thenon-natural amino acid is an insertion, appendage, or substitution foranother amino acid. 77. The peptide active agent conjugate or peptide ofembodiment 76, wherein the peptide is linked to the detectable agent atthe non-natural amino acid by a linker. 78. The peptide active agentconjugate or peptide of any one of embodiments 71-77, wherein the linkercomprises an amide bond, an ester bond, a carbamate bond, a hydrazonebond, an oxime bond, or a carbon-nitrogen bond. 79. The peptide activeagent conjugate or peptide of embodiment 74, wherein the cleavablelinker comprises a cleavage site for matrix metalloproteinases,thrombin, cathepsins, or beta-glucuronidase. 80. The peptide activeagent conjugate or peptide of any one of embodiments 71-74, wherein thepeptide is linked to the detectable agent via a stable linker. 81. Thepeptide active agent conjugate or peptide of any one of embodiments71-80, wherein the detectable agent is a fluorophore, a near-infrareddye, a contrast agent, a nanoparticle, a metal-containing nanoparticle,a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope,or a radionuclide chelator. 82. The peptide active agent conjugate orpeptide of any one of embodiments 71-81, wherein the detectable agent isa fluorescent dye. 83. A pharmaceutical composition comprising thepeptide active agent conjugate of any of the embodiments 1-3 or 5-82 ora salt thereof, or the peptide of any of embodiments 4-82 or a saltthereof, and a pharmaceutically acceptable carrier. 84. Thepharmaceutical composition of embodiment 83, wherein the pharmaceuticalcomposition is formulated for administration to a subject. 85. Thepharmaceutical composition of any of embodiments 83-84, wherein thepharmaceutical composition is formulated for inhalation, intranasaladministration, oral administration, topical administration, parenteraladministration, intravenous administration, subcutaneous administration,intra-articular administration, intramuscular administration,intraperitoneal administration, dermal administration, transdermaladministration, or a combination thereof. 86. A method of treating acondition in a subject in need thereof, the method comprisingadministering to the subject the peptide active agent conjugate of anyof embodiments 1-3 or 5-82, the peptide of any of embodiments 4-82, or apharmaceutical composition of any one of embodiments 83-85. 87. Themethod of embodiment 86, wherein the peptide active agent conjugate,peptide, or pharmaceutical composition is administered by inhalation,intranasally, orally, topically, parenterally, intravenously,subcutaneously, intra-articularly, intramuscularly administration,intraperitoneally, dermally, transdermally, or a combination thereof 88.The method any one of embodiments 86-87, wherein the peptide activeagent conjugate or the peptide homes, targets, or migrates to cartilageof the subject following administration. 89. The method of any ofembodiments 86-88, wherein the condition is associated with cartilage.90. The method of any one of embodiments 86-88, wherein the condition isassociated with a joint. 91. The method of any of embodiments 86-88,wherein the condition is an inflammation, a cancer, a degradation, agrowth disturbance, genetic, a tear, an infection, a disease, or aninjury. 92. The method of any of embodiments 86-88, wherein thecondition is a chondrodystrophy. 93. The method of any of embodiments86-88, wherein the condition is a traumatic rupture or detachment. 94.The method of any of embodiments 86-88, wherein the condition is acostochondritis. 95. The method of any of embodiments 86-88, wherein thecondition is a herniation. 96. The method of any of embodiments 86-88,wherein the condition is a polychondritis. 97. The method of any ofembodiments 86-88, wherein the condition is a chordoma. 98. The methodof any of embodiments 86-88, wherein the condition is a type ofarthritis. 99. The method of embodiment 98, wherein the type ofarthritis is rheumatoid arthritis. 100. The method of embodiment 198,wherein the type of arthritis is osteoarthritis. 101. The method of anyof embodiments 86-88, wherein the condition is achondroplasia. 102. Themethod of any of embodiments 86-88, wherein the condition is benignchondroma or malignant chondrosarcoma. 103. The method of any ofembodiments 86-88, wherein the condition is bursitis, tendinitis, gout,pseudogout, an arthropathy, psoriatic arthritis, ankylosing spondylitis,or an infection. 104. The method of embodiment 91, wherein the peptideactive agent conjugate, peptide, or pharmaceutical composition isadministered to treat the injury, to repair a tissue damaged by theinjury, or to treat a pain caused by the injury. 105. The method ofembodiment 91, wherein the peptide active agent conjugate, peptide, orpharmaceutical composition is administered to treat the tear or torepair a tissue damaged by the tear. 106. The method of any one ofembodiments 86-87, wherein the peptide active agent conjugate, peptide,or pharmaceutical composition homes, targets, or migrates to a kidney ofthe subject following administration. 107. The method of any one ofembodiments 86-87 or 106, wherein the condition is associated with akidney. 108. The method of embodiment 107, wherein the condition islupus nephritis, acute kidney injury (AKI), chronic kidney disease(CKD), hypertensive kidney damage, diabetic nephropathy, or renalfibrosis. 109. A method of imaging an organ or body region of a subject,the method comprising: administering to the subject the peptide activeagent conjugate of any of embodiments 1-3 or 5-82, peptide of any ofembodiments 4-82, or pharmaceutical composition of any one ofembodiments 83-85; and imaging the subject. 110. The method ofembodiment 109, wherein further comprising detecting a cancer ordiseased region, tissue, structure or cell. 111. The method of any oneof embodiments 109-110, further comprising performing surgery on thesubject. 112. The method of any one of embodiments 109-111, furthercomprising treating the cancer. 113. The method of any one ofembodiments 109-111, wherein the surgery comprises removing the canceror the diseased region, tissue, structure or cell of the subject. 114.The method of embodiment 111, further comprising imaging the cancer ordiseased region, tissue, structure, or cell of the subject aftersurgical removal. 115. The peptide active agent conjugate of any one ofembodiments 1-3 or 5-82, wherein the peptide active agent conjugate isexpressed as a fusion protein. 116. A compound having the structure ofFormula (I), or a pharmaceutically acceptable salt thereof:

wherein: R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, and R¹⁶ are eachindependently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH,sulfonate, —COOH, —SO₂—NH₂, C₁-C₆ alkoxy, alkylene-(C (═O))_(x)—,alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—; R⁹ ishydrogen, sulfonate, —COOH, alkylene-(C (═O))_(x)—, alkylene-(C(═O))_(x)—O—, or C₁-C₁₀ alkylene-(C (═O))_(x)—NR¹⁰—; L¹ is C₃-C₆alkylene; L² is C₁-C₁₀ alkylene; L³ is a bond, —O—, —NR¹⁰—, alkylene-,—O—NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-(O—C₁-C₆ alkylene)_(n)—, —NR¹⁰-L⁴—,—NR¹⁰—C₁-C₆ alkylene—NR¹¹—(C (═O) —C₁-C₆ alkylene-O—)_(m)—, or—NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆ alkylene—NR¹⁰—C₁-C₆ alkylene-; L⁴ is abond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-; R¹⁰ ishydrogen or C₁-C₆ alkyl; R¹¹ is hydrogen or C₁-C₆ alkyl; R¹² and R¹³ areeach independently selected from hydrogen, C₁-C₆ alkyl, or R¹² and R¹³are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring; R¹⁴ is hydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-aryl-A⁵,—(L⁵)—heteroaryl, —(L⁵)-heteroaryl-A⁵, —NR¹⁷ R¹⁸, R¹⁴ and R¹⁹ are joinedtogether along with the other atoms to which they are attached to form a5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴ andR²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring; L⁵ is a bond, C₁-C₁₀ alkylene, —O—, or —NR¹⁰—; R¹¹ and R¹⁸ areeach independently hydrogen or aryl; R¹⁹ and R²⁰ are each independentlyselected from hydrogen, C₁-C₆ alkyl, R¹⁴ and R¹⁹ are joined togetheralong with the other atoms to which they are attached to form a5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴ andR²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring; n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; p is 0, 1, 2, or 3; q is0, 1, 2, or 3; x is 0 or 1; and one of A¹, A², A³, A⁴, or A⁵ is apolypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof and theothers of A¹, A², A³, A⁴, or A⁵ are each independently absent, hydrogen,—COOH, or sulfonate. 117. The compound of embodiment 116 having thestructure of Formula (II), or a pharmaceutically acceptable saltthereof:

118. The compound of any one of embodiments 116 or 117 having thestructure of Formula (III), or a pharmaceutically acceptable saltthereof:

wherein: R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are eachindependently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH,sulfonate, —COOH, —SO₂—NH₂, or C₁-C₆ alkoxy; R⁹ is hydrogen, sulfonate,or —COOH; L¹ is C₃-C₆ alkylene; L² is C₁-C₁₀ alkylene; L³ is a bond,—O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-(O—C₁-C₆ alkylene_(n)—, —NR¹⁰-L⁴-, —NR¹⁰—C₁-C₆ alkylene-NR¹¹—(C (═O)—C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C₁-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-; L⁴ is a bond, -heterocyclyl- , or—heterocyclyl-C₁-C₆ alkylene-; R¹⁰ is hydrogen or C₁-C₆ alkyl; R¹¹ ishydrogen or C₁-C₆ alkyl; R¹² and R¹³ are independently selected fromhydrogen, C₁-C₆ alkyl, or R¹² and R¹³ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring; R¹⁴ is hydrogen or C₁-C₆alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷ R¹⁸, R¹⁴ and R¹⁹ arejoined together along with the other atoms to which they are attached toform a 5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴and R²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring; L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—; R¹⁷ and R¹⁸ are eachindependently hydrogen or aryl; R¹⁹ and R²⁰ are independently selectedfrom hydrogen, C₁-C₆ alkyl, R¹⁴ and R¹⁹ are joined together along withthe other atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring, or R¹⁴ and R²⁰ are joinedtogether along with the other atoms to which they are attached to form a5-membered or 6-membered carbocyclic or heterocyclic ring; n is 0, 1, 2,or 3; m is 0, 1, 2, or 3; p is 0, 1, 2, or 3; q is 0, 1, 2, or 3; and A⁴is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof 119. Thecompound of any one of embodiments 116 or 117 having the structure ofFormula (IV), or a pharmaceutically acceptable salt thereof:

wherein: R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are eachindependently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH,sulfonate, —COOH, —SO₂—NH₂, or C₁-C₆ alkoxy; R³ is selected from C₁-C₁₀alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C (═O))_(x)—O—, or C₁-C₁₀alkylene-(C (═O))_(x)—NR¹⁰—; R⁹ is hydrogen, sulfonate, or —COOH, orC₁-C₁₀ alkyl; L¹ is C₃-C₆ alkylene; L² is C₁-C₁₀ alkylene; L³ ishydrogen, sulfonate, —COOH, C₁-C₁₀ alkyl; L⁴ is a bond, -heterocyclyl- ,or -heterocyclyl- C₁-C₆ alkylene-; R¹⁰ is hydrogen or C₁-C₆ alkyl; R¹¹is hydrogen or C₁-C₆ alkyl; R¹² and R¹³ are independently selected fromhydrogen, C₁-C₆ alkyl, or R¹² and R¹³ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring; R¹⁴ is hydrogen or C₁-C₆alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷ R¹⁸, R¹⁴ and R¹⁹ arejoined together along with the other atoms to which they are attached toform a 5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴and R²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring; L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—; R¹⁷ and R¹⁸ are eachindependently hydrogen or aryl; R¹⁹ and R²⁰ are independently selectedfrom hydrogen, C₁-C₆ alkyl, R¹⁴ and R¹⁹ are joined together along withthe other atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring, or R¹⁴ and R²⁰ are joinedtogether along with the other atoms to which they are attached to form a5-membered or 6-membered carbocyclic or heterocyclic ring; n is 0, 1, 2,or 3; m is 0, 1, 2, or 3; p is 0, 1, 2, or 3; q is 0, 1, 2, or 3; x is 0or 1; and A¹ is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 120. Thecompound of any one of embodiments 116 or 117 having the structure ofFormula (V), or a pharmaceutically acceptable salt thereof:

wherein: R¹, R², R³, R⁴, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are eachindependently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH,sulfonate, —COOH, —SO₂—NH₂, or C₁-C₆ alkoxy; R⁵ is selected from C₁-C₁₀alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C (═O))_(x)—O—, or C₁-C₁₀alkylene-(C (═O))_(x)—NR¹⁰—; R⁹ is hydrogen, sulfonate, or —COOH, orC₁-C₁₀ alkyl; L¹ is C₃-C₆ alkylene; L² is C₁-C₁₀ alkylene; L³ ishydrogen, sulfonate, —COOH, or C₁-C₁₀ alkyl; L⁴ is a bond,-heterocyclyl-, or -heterocyclyl- C₁-C₆ alkylene-; R¹⁰ is hydrogen orC₁-C₆ alkyl; R¹¹ is hydrogen or C₁-C₆ alkyl; R¹² and R¹³ areindependently selected from hydrogen, C₁-C₆ alkyl, or R¹² and R¹³ arejoined together along with the other atoms to which they are attached toform a 5-membered or 6-membered carbocyclic or heterocyclic ring; R¹⁴ ishydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷ R¹⁸, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring; L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—; R¹⁷and R¹⁸ are each independently hydrogen or aryl; R¹⁹ and R²⁰ areindependently selected from hydrogen, C₁-C₆ alkyl, R¹⁴ and R¹⁹ arejoined together along with the other atoms to which they are attached toform a 5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴and R²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring; n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; p is 0, 1, 2, or 3; q is0, 1, 2, or 3; xis 0 or 1; and A² is a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof. 121. The compound of any one of embodiments 116 or 117having the structure of Formula (VI), or a pharmaceutically acceptablesalt thereof:

wherein: R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are eachindependently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH,sulfonate, —COOH, —SO₂—NH₂, or C₁-C₆ alkoxy; R⁹ is selected from C₁-C₁₀alkylene-(C (═O))_(x)—, C₁-C₁₀ alkylene-(C (═O))_(x)—O—, or C₁-C₁₀alkylene-(C (═O))_(x)—NR¹⁰—; L¹ is C₃-C₆ alkylene; L² is C₁-C₁₀alkylene; L³ is hydrogen, sulfonate, —COOH, or C₁-C₁₀ alkyl; L⁴ is abond, -heterocyclyl- , or -heterocyclyl- C₁-C₆ alkylene-; R¹⁰ ishydrogen or C₁-C₆ alkyl; R¹¹ is hydrogen or C₁-C₆ alkyl; R¹² and R¹³ areindependently selected from hydrogen, C₁-C₆ alkyl, or R¹² and R¹³ arejoined together along with the other atoms to which they are attached toform a 5-membered or 6-membered carbocyclic or heterocyclic ring; R¹⁴ ishydrogen or C₁-C₆ alkylene, -(L⁵)-aryl, -(L⁵)-heteroaryl, —NR¹⁷ R¹⁸, R¹⁴and R¹⁹ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring, or R¹⁴ and R²⁰ are joined together along with the other atoms towhich they are attached to form a 5-membered or 6-membered carbocyclicor heterocyclic ring; R¹⁷ and R¹⁸ are each independently hydrogen oraryl; R¹⁹ and R²⁰ are independently selected from hydrogen, C₁-C₆ alkyl,R¹⁴ and R¹⁹ are joined together along with the other atoms to which theyare attached to form a 5-membered or 6-membered carbocyclic orheterocyclic ring, or R¹⁴ and R²⁰ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring; n is 0, 1, 2, or 3; m is 0,1, 2, or 3; p is 0, 1, 2, or 3; q is 0, 1, 2, or 3; xis 0 or 1; L⁵ is abond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—; A³ is a polypeptide having at least85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof. 122. The compound of any one of embodiments 116 or 117having the structure of Formula (III), or a pharmaceutically acceptablesalt thereof:

wherein: R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ , R¹⁵, and R¹⁶ are eachindependently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkylene-COOH,sulfonate, —COOH, —SO₂—NH₂, or C₁-C₆ alkoxy; R⁹ is hydrogen, sulfonate,or —COOH; L¹ is C₃-C₆ alkylene; L² is C₁-C₁₀ alkylene; L³ is a bond,—O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, —O—NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-(O—C₁-C₆ lkylene)_(n)-, —NR¹⁰-L⁴—, —NR¹⁰—C₁-C₆ alkylene-NR¹¹—(C (═O)—C₁-C₆ alkylene-O—)_(m)—, or —NR¹⁰—C₁-C₆ alkylene-NR¹⁰—C1-C₆alkylene-NR¹⁰—C₁-C₆ alkylene-; L⁴ is a bond, -heterocyclyl- , or-heterocyclyl-C₁-C₆ alkylene-; R¹⁰ is hydrogen or C₁-C₆ alkyl; R¹¹ ishydrogen or C₁-C₆ alkyl; R¹² and R¹³ are independently selected fromhydrogen, C₁-C₆ alkyl, or R¹² and R¹³ are joined together along with theother atoms to which they are attached to form a 5-membered or6-membered carbocyclic or heterocyclic ring; R¹⁴ is -(L⁵)-aryl-A⁵, or-(L⁵)-heteroaryl-A⁵; L⁵ is a bond, C₁-C₁₀ alkylene, —O—, —NR¹⁰—; R¹⁷ andR¹⁸ are each independently hydrogen or aryl; R¹⁹ and R²⁰ areindependently selected from hydrogen, C₁-C₆ alkyl, R¹⁴ and R¹⁹ arejoined together along with the other atoms to which they are attached toform a 5-membered or 6-membered carbocyclic or heterocyclic ring, or R¹⁴and R²⁰ are joined together along with the other atoms to which they areattached to form a 5-membered or 6-membered carbocyclic or heterocyclicring; n is 0, 1, 2, or 3; m is 0, 1, 2, or 3; p is 0, 1, 2, or 3; q is0, 1, 2, or 3; x is 0 or 1; A⁴ is hydrogen, —COOH, or sulfonate; and A⁵is a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 123. Thecompound of any one of embodiments 116-122, wherein A¹, A², and A³ areabsent. 124. The compound of any one of embodiments 116-121, wherein A⁵is hydrogen. 125. The compound of any one of embodiments 116-124,wherein R³, R⁴, R⁵, and R⁶ are each independently C₁-C₆ alkyl. 126. Thecompound of any one of embodiments 116-124, wherein R³, R⁴, R⁵, R⁶ areeach independently methyl. 127. The compound of any one of embodiments116-126, wherein R¹, R², R⁷, R⁸, R¹⁵, and R¹⁶ are each independentlyselected from hydrogen or sulfonate. 128. The compound of any one ofembodiments 116-126, wherein R¹, R², R⁷, R⁸, R¹⁵, and R¹⁶ are eachindependently hydrogen. 129. The compound of any one of embodiments116-128, wherein R¹², R¹³, R¹⁴, R¹⁹, R²⁰ are each independentlyhydrogen. 130. The compound of any one of embodiments 116-129, whereinR¹² and R¹³ join together along with the atoms to which they areattached to form a six-membered carbocyclic ring. 131. The compound ofany one of embodiments 116-130, wherein R¹² and R¹³ join together alongwith the atoms to which they are attached to form a five-memberedcarbocyclic ring. 132. The compound of any one of embodiments 116-131,wherein R¹⁴ and R¹⁹ join together along with the atoms to which they areattached to form a six-membered carbocyclic ring. 133. The compound ofany one of embodiments 116-132, wherein R¹⁴ and R²⁰ join together alongwith the atoms to which they are attached to form a six-memberedcarbocyclic ring. 134. The compound of any one of embodiments 116-133,wherein L¹ is C₃-C₆ alkylene. 135. The compound of any one ofembodiments 116-133, wherein L¹ is C3-Cs alkylene 136. The compound ofany one of embodiments 116-133, wherein L¹ is propylene. 137. Thecompound of any one of embodiments 116-133, wherein L¹ is butylene. 138.The compound of any one of embodiments 116-133, wherein L¹ is pentylene.139. The compound of any one of embodiments 116-138, wherein L² is C₃-C₆alkylene. 140. The compound of any one of embodiments 116-138, whereinL² is propylene. 141. The compound of any one of embodiments 116-138,wherein L² is butylene. 142. The compound of any one of embodiments116-138, wherein L² is pentylene. 143. The compound of any one ofembodiments 116-142, wherein R⁹ is sulfonate. 144. The compound of anyone of embodiments 116-142, wherein R⁹ is hydrogen. 145. The compound ofany one of embodiments 116-144, wherein R¹⁴ is hydrogen. 146. Thecompound of any one of embodiments 116-144, wherein R¹⁴ is -(L⁵)-aryl.147. The compound of any one of embodiments 116-144, wherein R¹⁴ is-(L⁵)-aryl-A⁵. 148. The compound of any one of embodiments 116-146,wherein R¹ is hydrogen. 149. The compound of any one of embodiments116-147, wherein R² is hydrogen. 150. The compound of any one ofembodiments 116-148, wherein R³ is methyl. 151. The compound of any oneof embodiments 116-149, wherein R⁴ is methyl. 152. The compound of anyone of embodiments 116-150, wherein R⁵ is methyl. 153. The compound ofany one of embodiments 116-152, wherein R⁶ is methyl. 154. The compoundof any one of embodiments 116-153, wherein R⁷ is hydrogen. 155. Thecompound of any one of embodiments 116-154, wherein R⁸ is hydrogen. 156.The compound of any one of embodiments 116-155, wherein R¹² is hydrogen.157. The compound of any one of embodiments 116-156, wherein R¹³ ishydrogen. 158. The compound of any one of embodiments 116-157, whereinR¹⁴ is hydrogen. 159. The compound of any one of embodiments 116-158,wherein R¹⁹ is hydrogen. 160. The compound of any one of embodiments116-159, wherein R²⁰ is hydrogen. 161. The compound of any one ofembodiments 116-160, wherein R¹⁷ and R¹⁸ are independently phenyl. 162.The compound of any one of embodiments 116-161, wherein L³ is selectedfrom a bond, —O—, —NR¹⁰—, —NR¹⁰—C₁-C₆ alkylene-, or —O—NR¹⁰—, or—NR¹⁰-L⁴-. 163. The compound of any one of embodiments 116-162, whereinL³ is a bond. 164. The compound of any one of embodiments 116-163,wherein R¹⁰ is hydrogen. 165. The compound of any one of embodiments116-164, wherein L⁴ is -heterocyclyl- or -heterocyclyl-C₁-C₆ alkylene-.166. The compound of any one of embodiments 116-165, wherein L⁴ is-piperizinyl-(C₁-C₆ alkylene)-. 167. The compound of any one ofembodiments 116-166, wherein L⁴ is

168. The compound of any one of embodiments 116-167, wherein R¹¹ ishydrogen. 169. The compound of any one of embodiments 116-168, wherein pis 1. 170. The compound of any one of embodiments 116-169, wherein qis 1. 171. The compound of any one of embodiments 116 or 117 having thestructure of any one of

Formulas (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV):

172. The compound of any one of embodiments 116-170, wherein one of A¹,A², A³, A⁴, or A⁵ is a polypeptide having at least 87% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 173.The compound of any one of embodiments 116-170, wherein one of A¹, A²,A³, A⁴, or A⁵ is a polypeptide having at least 90% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 174.The compound of any one of embodiments 116-170, wherein one of A¹, A²,A³, A⁴, or A⁵ is a polypeptide having at least 92% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 175.The compound of any one of embodiments 116-170, wherein one of A¹, A²,A³, A⁴, or A⁵ is a polypeptide having at least 95% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 176.The compound of any one of embodiments 116-170, wherein one of A¹, A²,A³, A⁴, or A⁵ is a polypeptide having at least 97% sequence identitywith MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 177.The compound of any one of embodiments 116-170, wherein one of A¹, A²,A³, A⁴, or A⁵ is a polypeptide having 100% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof. 178. Thecompound of any one of embodiments 116-170, wherein one of A¹, A², A³,A⁴, or A⁵ is a polypeptide having the sequenceMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof 179. Thecompound of any one of embodiments 116-178, wherein the fragment of A¹,A², A³, A⁴, or A⁵ has a length of at least 25 amino acid residues. 180.The compound of any one of embodiments 116-178, wherein the fragment ofA¹, A², A³, A⁴, or A⁵ has a length of at least 27 amino acid residues.181. The compound of any one of embodiments 116-178, wherein thefragment of A¹, A², A³, A⁴, or A⁵ has a length of at least 29 amino acidresidues. 182. The compound of any one of embodiments 116-178, whereinthe fragment of A¹, A², A³, A⁴, or A⁵ has a length of at least 31 aminoacid residues. 183. The compound of any one of embodiments 116-178,wherein the fragment of A¹, A², A³, A⁴, or A⁵ has a length of at least33 amino acid residues. 184. The compound of any one of embodiments116-183, wherein one of A¹, A², A³, A⁴, or A⁵ is a a polypeptide havingat least 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCRor a fragment thereof having the tumor cell binding affinity of nativechlorotoxin. 185. The compound of any one of embodiments 116-183,wherein one of A¹, A², A³, A⁴, or A⁵ is a a polypeptide having at least85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof having essentially the same the tumor cell bindingaffinity of native chlorotoxin. 186. The compound of any one ofembodiments 116-185, wherein one of A¹, A², A³, A⁴, or A⁵ is a apolypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof having thetumor cell binding affinity of native chlorotoxinwherein one of A¹, A²,A³, A⁴, or A⁵ has a sequence selected from SEQ ID NOS: 1-481. 187. Thecompound of any one of embodiments 116-186, wherein the polypeptidecomprises at least one lysine amino acid residue. 188. The compound ofany one of embodiments 116-187, wherein the polypeptide comprises asingle lysine amino acid residue. 189. The compound of any one ofembodiments 116-187, wherein the polypeptide comprises one, two, orthree lysine amino acid residues. 190. The compound of any one ofembodiments 116-189, wherein the polypeptide comprises a lysine residueat the position corresponding to K-27 of native chlorotoxin. 191. Thecompound of any one of embodiments 116-190, wherein the polypeptidecomprises a lysine residue at the position corresponding to K-23 ofnative chlorotoxin. 192. The compound of any one of embodiments 116-191,wherein the polypeptide comprises a lysine residue at the positioncorresponding to K-15 of native chlorotoxin. 193. The compound of anyone of embodiments 116-192, wherein one or more of the amino acids ofthe polypeptide is substituted with a non-naturally occurring amino acidresidue. 194. The compound of any one of embodiments 116-193, wherein alysine is replaced with a nonnaturally occuring amino acid. 195. Thecompound of embodiment 194, wherein the non-naturally occurring aminoacid residue is a citrulline amno acid residue. 196. The compound ofembodiment 195, wherein L³ is attached to A⁴ at a citrulline amino acidresidue of the polypeptide. 197. The compound of any one of embodiments116-196, wherein L³ is attached to A⁴ at a lysine amino acid residue ofthe polypeptide. 198. The compound of any one of embodiments 116-196,wherein L³ is attached to A⁴ at the N-terminus of the polypeptide. 199.The compound of any one of embodiments 116-196, wherein L³ is attachedto A⁴ at the C-terminus of the polypeptide. 200. The compound of any oneof embodiments 116-196, wherein the R³ is attached to A¹ at a lysineamino acid residue of the peptide, a citrulline amino acid residue ofthe polypeptide, the N-terminus of the polypeptide, or the C-terminus ofthe polypeptide. 201. The compound of any one of embodiments 116-196,wherein the R⁵ is attached to A² at a lysine amino acid residue of thepolypeptide, a citrulline amino acid residue of the polypeptide, theN-terminus of the polypeptide, or the C-terminus of the polypeptide.202. The compound of any one of embodiments 116-196, wherein the R⁹ isattached to A³ at a lysine amino acid residue of the polypeptide, acitrulline amino acid residue of the polypeptide, the N-terminus of thepolypeptide, or the C-terminus of the polypeptide. 203. The compound ofany one of embodiments 116-196, wherein the aryl is attached to A⁵ at alysine amino acid residue of the polypeptide, a citrulline amino acidresidue of the polypeptide, the N-terminus of the polypeptide, or theC-terminus of the polypeptide. 204. The compound of any one ofembodiments 116-203, having the structure of any one of compounds 1 to720 as found in Tables 2-13. 205. The compound of any one of embodiments116-204, wherein the compound is conjugated to polyethylene glycol(PEG), hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer,a zwitterionic water soluble polymer, a water soluble poly(amino acid),an albumin derivative, or a fatty acid. 206. The compound of any one ofembodiments 116-205, wherein the polypeptide has an isoelectric point offrom 7.5 to 9.0. 207. The compound of any one of embodiments 116-206,wherein the polypeptide has an isoelectric point of from 8.0 to 9.0.208. The compound of any one of embodiments 116-205, wherein thepolypeptide has an isoelectric point of from 8.5 to 9.0. 209. Thecompound of any one of embodiments 116-208, wherein the polypeptidecomprises at least eight cysteine amino acid residues. 210. The compoundof any one of embodiments 116-208, wherein the polypeptide compriseseight cysteine amino acid residues. 211. The compound of any one ofembodiments 116-210, wherein the polypeptide comprises four disulfidebonds. 212. The compound of any one of embodiments 116-208, wherein thepolypeptide comprises from six to seven cysteine amino acid residues.213. The compound of any one of embodiments 116-208, wherein thepolypeptide comprises three disulfide bonds. 214. The compound of anyone of embodiments 116-213, wherein the polypeptide is basic and has anisoelectric point of greater than 7.5. 215. The compound of any one ofembodiments 116-214, wherein the spacing between the cysteine amino acidresidues in the polypeptide is essentially the same as in nativechlorotoxin. 216. The compound of any one of embodiments 116-215,wherein the distribution of charge on the surface of the polypeptide isessentially the same as in native chlorotoxin. 217. The compound of anyone of embodiments 116-216, wherein one or more of the methionine amioacid residies is replaced with an amino acid residue selected fromisoleucine, threonine, valine, leucine, serine, glycine, alanine, or acombination thereof. 218. The compound of any one of embodiments116-217, wherein the compound is capable of passing across the bloodbrain barrier. 219. The compound of any one of embodiments 116-218,further comprising a therapeutic agent attached to A. 220. The compoundof embodiment 219, wherein the therapeutic agent is a cytotoxic agent.221. A composition comprising the compound of any one of embodiments116-220 and a pharmaceutically acceptable carrier. 222. The compositionof embodiment 221, wherein the composition is formulated for parenteraladministration. 223. The composition of any one of embodiments 221-222,wherein the composition is formulated for intravenous administration,intramuscular administration, subcutaneous administration, or acombination thereof 224. The composition of any one of embodiments221-223, wherein the pharmaceutically acceptable carrier comprises anosmolyte. 225. The composition of embodiments 224, wherein the osmolytecomprises a sugar, a sugar alcohol, or a combination thereof. 226. Thecomposition of embodiment 225, wherein the sugar alcohol is selectedfrom sorbitol, inositol, mannitol, xylitol and glycerol, or acombination thereof 227. The composition of any one of embodiments225-226, wherein the sugar alcohol comprises mannitol. 228. Thecomposition of any one of embodiments 226-227, comprising from 2% to 20%(wt/vol %) mannitol. 229. The composition of any one of embodiments226-227, comprising from 2% to 10% (wt/vol %) mannitol. 230. Thecomposition of any one of embodiments 226-227, comprising essentially 5%(wt/vol %) mannitol. 231. The composition of embodiment 225, wherein thesugar is selected from trehalose, lactose, sucrose, glucose, galactose,maltose, mannose, fructose, dextrose, or a combination thereof 232. Thecomposition of embodiment 225, wherein the sugar is selected fromtrehalose, sucrose, or a combination thereof 233. The composition of anyone of embodiments 231-232, comprising from 1% to 40% (wt/vol %) oftrehalose, sucrose, or a combination of trehalose and sucrose. 234. Thecomposition of any one of embodiments 231-232, comprising from 1% to 20%(wt/vol %) of trehalose, sucrose, or a combination of trehalose andsucrose. 235. The composition of any one of embodiments 231-232,comprising 2% (wt/vol %) of trehalose, sucrose, or a combination oftrehalose and sucrose. 236. The composition of embodiment 211, whereinthe osmolyte is selected from glycine, carnitine, ethanolamine, theirphosphates, mono sugars, or a combination thereof 237. The compositionof any one of embodiments 221-236, wherein the composition is isotonic.238. The composition of any one of embodiments 221-236, wherein thecomposition is essentially isotonic. 239. The composition of any one ofembodiments 221-238, wherein the ionic strength of the composition isless than 50 mM. 240. The composition of any one of embodiments 221-238,wherein the ionic strength of the composition is less than 10 mM. 241.The composition of any one of embodiments 221-240, wherein thepharmaceutically acceptable carrier comprises a buffer. 242. Thecomposition of embodiment 241, wherein the buffer is selected from tris,HEPES, histidine, ethylene diamine, or a combination thereof. 243. Thecomposition of embodiment 241, wherein the buffer is selected from tris,histidine, or a combination thereof. 244. The composition of embodiment241, wherein the buffer comprises histidine. 245. The composition ofembodiment 244, wherein histidine is L-histidine. 246. The compositionof any one of embodiments 244-245, comprising at least 100 mM histidine.247. The composition of any one of embodiments 244-245, comprising atleast 50 mM histidine. 248. The composition of any one of embodiments244-245, comprising at least 20 mM histidine. 249. The composition ofany one of embodiments 244-245, comprising 10 to 100 mM histidine. 250.The composition of any one of embodiments 244-245, comprising 10 to 20mM histidine. 251. The composition of any one of embodiments 221-250,wherein the pharmaceutically acceptable carrier comprises anantioxidant, a free radical scavenger, a quencher, an antioxidantsynergist or a combination thereof. 252. The composition of embodiment251, wherein the antioxidant is selected from methionine, butylatedhydroxytoluene, butylated hydroxyanisole, propyl gallate, or acombination thereof. 253. The composition of embodiment 252, wherein theantioxidant comprises methionine. 254. The composition of embodiment253, wherein the methionine is L-methionine. 255. The composition of anyone of embodiments 252-254, comprising at least 20 mM methionine. 256.The composition of any one of embodiments 252-254, comprising at least10 mM methionine. 257. The composition of any one of embodiments221-256, wherein the pharmaceutically acceptable carrier comprises asurfactant. 258. The composition of embodiment 257, wherein thesurfactant is selected from polysorbate 20, polysorbate 80, a pluronic,polyoxyethylene sorbitan mono-oleate, polyethylene mono-laureate,N-actylglucoside, or a combination thereof. 259. The composition ofembodiment 257, wherein the surfactant is polysorbate 20. 260. Thecomposition of any one of embodiments 258-259, comprising from 0.0001%to 0.1% (wt/vol %) polysorbate 20. 261. The composition of any one ofembodiments 221-260, wherein the pharmaceutically acceptable carriercomprises a cyclodextrin. 262. The composition of embodiment 261,wherein the cyclodextrin is (2-hydroxypropyl)-β-cyclodextrin. 263. Thecomposition of any one of embodiments 221-262, wherein thepharmaceutically acceptable carrier comprises a reconstitutionstabilizer. 264. The composition of embodiment 263, wherein thereconstitution stabilizer comprises a water-soluble polymer. 265. Thecomposition of embodiment 264, wherein the water-soluble polymer isselected from a polaxamer, a polyol, a polyethylene glycol, apolyvinylalcohol, a hydroxyethyl starch, dextran, polyvinylpyrrolidenepoly(acrylic acid), or a combination thereof 266. The composition of anyone of embodiments 221-265, further comprising a metal chelator. 267.The composition of embodiment 266, wherein the metal chelator isselected from EDTA, deferoxamine mesylate, EGTA, fumaric acid, and malicacid, salts thereof, or combinations thereof 268. The composition of anyone of embodiments 266-267, wherein the metal chelator comprises EDTA orsalts thereof 269. The composition of embodiment 153, having an EDTAconcentration of about 0.0001 mg/ml to about 0.01 mg/ml. 270. Thecomposition of any one of embodiments 221-269, having a pH of from 6 to7.5. 271. The composition of any one of embodiments 221-256, having a pHof from 6.5 to 7.0. 272. The composition of any one of embodiments221-270, wherein the concentration of the compound is from 1 mg/mL to 40mg/mL. 273. The composition of any one of embodiments 221-270, whereinthe concentration of the compound is from 1 mg/mL to 20 mg/mL. 274. Thecomposition of any one of embodiments 221-270, wherein the concentrationof the compound is from 4 mg/mL to 10 mg/mL. 275. The composition of anyone of embodiments 221-270, wherein the concentration of the compound isfrom 5 mg/mL to 8 mg/mL. 276. The composition of any one of embodiments221-270, wherein the concentration of the compound is from 5 mg/mL to 6mg/mL. 277. The composition of any one of embodiments 221-276, whereinthe pharmaceutically acceptable carrier comprises tris, D-mannitol, anda pH of essentially 6.8. 278. The composition of any one of embodiments221-276, wherein the pharmaceutically acceptable carrier consistsessentially of tris, D-mannitol, and a pH of 6.8. 279. The compositionof any one of embodiments 221-276, wherein the pharmaceuticallyacceptable carrier comprises L-histidine, D-mannitol, L-methionine, anda pH of essentially 6.8. 280. The composition of any one of embodiments221-276, wherein the pharmaceutically acceptable carrier consistsessentially of L-histidine, D-mannitol, L-methionine, and a pH of 6.8.281. The composition of any one of embodiments 221-276, wherein thepharmaceutically acceptable carrier comprises L-histidine, D-mannitol,polysorbate 20, and a pH of essentially 6.8. 282. The composition of anyone of embodiments 221-276, wherein the pharmaceutically acceptablecarrier comprises L-histidine, D-mannitol, and a pH of essentially 6.8.283. The composition of any one of embodiments 221-276, wherein thepharmaceutically acceptable carrier comprises L-histidine, D-mannitol,polysorbate 20, trehalose, and a pH of essentially 6.8. 284. Thecomposition any one of embodiments 221-283, wherein the composition islyophilized. 285. A method of producing the composition of embodiment284, the method comprising: providing the composition; and yophilizingthe composition, thereby producing the lyophilized composition. 286. Thecomposition of any one of embodiments 221-285, wherein the compositionis reconstituted from a lyophilized form. 287. A method of producing thecomposition of embodiment 286, the method comprising: providing thelyophilized composition of embodiment 284; and reconstituting thecomposition with a solution to produce a reconstituted composition. 288.The composition of embodiment 286, wherein the solution comprises water.289. A composition comprising a polypeptide conjugated to a fluorescentdye and histidine. 290. The composition of embodiment 289, wherein thehistidine is L-histidine. 291. The composition of any one of embodiments289-290, comprising at least 50 mM histidine. 292. The composition ofany one of embodiments 289-290, comprising at least 20 mM histidine.293. The composition of any one of embodiments 289-290, comprising 10 to100 mM histidine. 294. The composition of any one of embodiments289-290, comprising 10 to 20 mM histidine. 295. The composition of anyone of embodiments 289-294, wherein the polypeptide conjugated to thefluorescent dye is a compound of any one of embodiments 116-220. 296. Akit comprising: a vessel configured to contain a fluid; the compositionof any one of embodiments 221-295 comprised within the vessel; and anelastomeric closure affixed to the vessel. 297. The kit of embodiment296, further comprising a light shield. 298. The kit of embodiment 297,wherein the light shield is a physical barrier configured to block atleast a portion of the light incident on the vessel from thecomposition. 299. The kit of embodiment 298, wherein the physicalbarrier comprises an opaque or semi-opaque material. 300. The kit of anyone of embodiments 296-299, wherein the vessel is a glass vial. 301. Thekit of embodiment 300, wherein the glass vial comprises clear or amberglass. 302. The kit of any one of embodiments 296-301, wherein the glassvial is an untreated glass container. 303. The kit of any one ofembodiments 300-302, wherein the glass vial comprises USP Type I, TypeII, Type III, or Type IV glass. 304. The kit of any one of embodiments296-303, wherein the inner portion of the vessel further comprises asilica (Si02) coating or silicone coating. 305. The kit of any one ofembodiments 302-304, wherein the untreated glass container is selectedfrom an ampoule, vial, ready-to-use syringe, or carpoule. 306. The kitof any one of embodiments 296-305, wherein the elastomeric closure is ahalobutyl rubber closure. 307. The kit of embodiment 306, wherein thehalobutyl rubber closure is selected from a chlorobutyl rubber closureor a bromobutyl rubber closure. 308. The kit of any one of embodiments296-307, wherein the elastomeric closure is coated with Fluorotec, B2,or a combination thereof 309. The kit of any one of embodiments 296-308,further comprising an opaque secondary package surrounding the vessel.310. The kit of embodiment 309, wherein the opaque secondary packagecomprises an opaque box, an opaque aluminum foil pouch, or a combinationthereof 311. The kit of any one of embodiments 309-310, wherein theopaque secondary package is configured to block at least 90% of thelight incident on the package exterior from the composition. 312. Thekit of any one of embodiments 309-310, wherein the opaque secondarypackage is configured to block at least 95% of the light incident on thepackage exterior from the composition. 313. The kit of any one ofembodiments 309-310, wherein the opaque secondary package is configuredto block at least 99% of the light incident on the package exterior fromthe composition. 314. The kit of any one of embodiments 309-310, whereinthe opaque secondary package is configured to block at least 99.9% ofthe light incident on the package exterior from the composition. 315.The kit of any one of embodiments 296-314, wherein the vessel comprisesa reduced-oxygen environment in contact with the composition. 316. Thekit of any one of embodiments 296-315, wherein the vessel comprises aninert gas in contact with the composition. 317. The kit of any one ofembodiments 315-316, wherein the composition is sparged with an inertgas, thereby producing the reduced-oxygen environment in the vessel.318. The kit of any one of embodiments 201 to 202, wherein the inert gascomprises nitrogen or argon. 319. A composition comprising a compoundcomprising a polypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof, wherein whenthe composition is intravenously administering to a human subject at adose of from 1 mg to 30 mg, the composition produces in the humansubject an average maximum compound blood plasma concentration (averageC_(max))of at least from 110 ng/mL to240 ng/mL per each 1 mg dosage ofthe compound administered. 320. The composition of embodiment 303,wherein the compound comprises the compound of any one of embodiments116-120. 321. The composition of any one of embodiments 319-320, whereinthe composition comprises the composition of any one of embodiments221-295. 322. A method of administering a composition to a humansubject, the method comprising: intravenously administering to the humansubject a dose of from 1 mg to 30 mg of a compound comprising apolypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof; andproducing in the human subject an average maximum compound blood plasmaconcentration (average C_(max))of at least from 110 ng/mL to240 ng/mLper each 1 mg dosage of the compound administered. 323. A method ofdetecting a cancer cell in a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof conjugated to a detectable label; producing in thehuman subject an average maximum compound blood plasma concentration(average C_(max))of at least from 110 ng/mL to 240 ng/mL per each 1 mgdosage of the compound administered; and detecting the presence orabsence of the detectable label in the human subject, wherein thepresence of the detectable label indicates the presence of the cancercell. 324. A method of diagnosing cancer in a human subject, the methodcomprising: intravenously administering to the human subject a dose offrom 1 mg to 30 mg of a compound comprising a polypeptide having atleast 85% sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR ora fragment thereof conjugated to a detectable label; producing in thehuman subject an average maximum compound blood plasma concentration(average C_(max))of at least from 110 ng/mL to 240 ng/mL per each 1 mgdosage of the compound administered; and detecting the presence orabsence of the detectable label in the human subject, wherein thepresence of the detectable label indicates a diagnosis of cancer. 325. Amethod of treating cancer in a human subject, the method comprising:intravenously administering to the human subject a dose of from 1 mg to30 mg of a compound comprising a polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or afragment thereof conjugated to a therapeutic agent; producing in thehuman subject an average maximum compound blood plasma concentration(average C_(max))of at least from 110 ng/mL to 240 ng/mL per each 1 mgdosage of the compound administered; and reducing or improving a symptomor condition associated with cancer in the human subject. 326. Themethod of embodiment 325, wherein the human subject is in need thereof.327. The method of any one of embodiments 325-326, comprisingadministering a therapeutically effective dose of the compound to thehuman subject. 328. A method of administering a composition to a humansubject, the method comprising: intravenously administering to the humansubject a dose of from 1 mg to 30 mg of a compound comprising apolypeptide having at least 85% sequence identity withMCMPCFTTDHQMARRCDDCCGGRGRGKCYGPQCLCR or a fragment thereof; andproducing in the human subject pharmacokinetic profile of FIG. 27 . 329.A method of administering a composition to a human subject, the methodcomprising: intravenously administering to the human subject a dose offrom 1 mg to 30 mg of a compound of any one of embodiments 116-220; andproducing in the human subject an average maximum compound blood plasmaconcentration (average C_(max))of at least from 110 ng/mL to240 ng/mLper each 1 mg dosage of the compound administered. 330. The method ofany one of embodiments 322-329, wherein the average time (averageT_(max)) at which the average C_(max) is reached is at 5±4 minutesfollowing administration of the compound. 331. The method of any one ofembodiments 322-330, wherein the average time (average T₇₅) at which theaverage compound blood plasma concentration reaches 75% of the averageC_(max) (average C75) is reached is at 8±5 minutes followingadministration of the compound. 332. The method of any one ofembodiments 322-331, wherein the average time (average T₅₀) at which theaverage compound blood plasma concentration reaches 50% of the averageC_(max) (average C50) is reached is at 20±8 minutes followingadministration of the compound. 333. The method of any one ofembodiments 322-332, wherein the average time (average T₂₅) at which theaverage compound blood plasma concentration reaches 25% of the averageC_(max) (average C25) is reached is at 30±12 minutes followingadministration of the compound. 334. The method of any one ofembodiments 322-333, further comprising producing in the human subjectan average chlorotoxin polypeptide plasma area under the curve (averageAUC) of from 50 hr*ng/mL to 120 hr*ng/mL per each 1 mg dosage ofchlorotoxin polypeptide administered. 335. The method of any one ofembodiments 334-334, further comprising producing in the human subjectan average chlorotoxin polypeptide plasma area under the curve (averageAUC) of from 60 hr*ng/mL to 110 hr*ng/mL per each 1 mg dosage ofchlorotoxin polypeptide administered. 336. The method of embodiment 335,wherein 75% of the average AUC occurs within 40±15 minutes afteradministering the compound. 337. The method of any one of embodiments335-336, wherein 50% of the average AUC occurs within 21±8 minutes afteradministering the compound. 338. The method of any one of embodiments335-337, wherein 25% of the average AUC occurs within 9±5 minutes afteradministering the compound. 339. The method of any one of embodiments322-338, wherein the compound comprises the compound of any one ofembodiments 116-220. 340. The method of any one of embodiments 207 to223, wherein the composition comprises the composition of any one ofembodiments 221-295. 341. A method for detecting a cancer cell in asubject, the method comprising: administering to the subject thecompound of any one of embodiments 116-220; and detecting the presenceor absence of the compound in the subject, wherein the presence of thecompound indicates the presence of a cancer cell. 342. The method ofembodiment 341, further comprising administering the compound as a partof a composition of any one of embodiments 221-295. 343. The method ofany one of embodiments 322 and 342, wherein the cancer is selected fromglioma, astrocytoma, medulloblastoma, choroids plexus carcinoma,ependymoma, brain tumor, neuroblastoma, adenocarcinoma, basal cellcarcinoma, squamous cell carcinoma, head and neck cancer, lung cancer,breast cancer, intestinal cancer, pancreatic cancer, liver cancer,kidney cancer, sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma,carcinoma, melanoma, ovarian cancer, cervical cancer, lymphoma, thyroidcancer, anal cancer, colo-rectal cancer, endometrial cancer, germ celltumor, laryngeal cancer, multiple myeloma, prostate cancer,retinoblastoma, gastric cancer, testicular cancer, or Wilm's tumor. 344.The method of any one of embodiments 332 and 342, wherein the cancer isselected from glioma, medulloblastoma, sarcoma, breast cancer, lungcancer, prostate cancer, or intestinal cancer. 345. The method of anyone of embodiments 322-344, wherein the cancer cell expresses a site towhich native chlorotoxin binds. 346. The method of any one ofembodiments 322-345, comprising detecting the compound by fluorescenceimaging. 347. The method of any one of embodiments 322-345, furthercomprising differentiating a focus of a cancer that expresses a site towhich native chlorotoxin binds from non-neoplastic tissue. 348. Themethod of any one of embodiments 322-347, further comprising surgicallyremoving from the subject a cancer cell that is detected. 349. Themethod of any one of embodiments 322-348, further comprising determiningthe location of a cancer cell in the subject before surgically removingthe cancer cell from the subject, during surgical removal of the cancercell from the subject, after removing the cancer cell from the subject,or a combination thereof 350. The method of any one of embodiments322-349, wherein the compound binds to the cancer cell. 351. The methodof any one of embodiments 322-350, wherein the subject is a humansubject. 352. The method of any one of embodiments 323-351, wherein thedetection is performed in vivo or ex vivo. 353. A method ofadministering the compound of any one of embodiments 116-220 to asubject, the method comprising administering a therapeutically effectiveamount of the compound to the subject. 354. The method of any one ofembodiments 322-353, wherein the subject is in need thereof 355. Themethod of any one of embodiments 353-354, wherein a therapeuticallyeffective amount is a dosage sufficient for the detection of a cancercell in the subject. 356. The method of any one of embodiments 322-355,wherein the dosage is from 0.1 mg to 100 mg. 357. The method of any oneof embodiments 322-355, wherein the dosage is from 1 mg to 30 mg. 358.The method of any one of embodiments 322-355, wherein the dosage is from3 mg to 30 mg. 359. A method of treating a subject in need thereof, themethod comprising administering to the subject the composition of anyone of embodiments 219-220 in an amount sufficient to treat cancer inthe subject. 360. The method of embodiment 359, wherein the cancer isselected from glioma, astrocytoma, medulloblastoma, choroids plexuscarcinoma, ependymoma, brain tumor, neuroblastoma, head and neck cancer,lung cancer, breast cancer, intestinal cancer, pancreatic cancer, livercancer, kidney cancer, sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing'ssarcoma, carcinoma, melanoma, ovarian cancer, cervical cancer, lymphoma,thyroid cancer, anal cancer, colo-rectal cancer, endometrial cancer,germ cell tumor, laryngeal cancer, multiple myeloma, prostate cancer,retinoblastoma, gastric cancer, testicular cancer, or Wilm's tumor. 361.The method of embodiment 359, wherein the cancer cell is selected fromglioma, medulloblastoma, sarcoma, prostate cancer, or intestinal cancer.362. The method of any one of embodiments 322-361, wherein the cancercell expresses a site to which native chlorotoxin binds. 363. The methodof embodiment 362, wherein the binding is selective. 364. The method ofany one of embodiments 322-363, wherein the compound is administeredparenterally. 365. The method of any one of embodiments 341-364, whereinthe compound is administered intravenously. 366. The method of any oneof embodiments 341-364, wherein the compound is administeredsubcutaneously. 367. A method of detecting soft-tissue sarcoma in anindividual, comprising the steps of: a) administering a chlorotoxinconjugate to the individual, wherein the chlorotoxin conjugate binds tothe soft-tissue sarcoma; and b) imaging, visualizing, or analyzing thebound chlorotoxin conjugate. 368. The method of embodiment 367, whereinthe detecting comprises in vivo, or ex vivo detection. 369. The methodof embodiment 367, wherein the imaging, visualizing, or analyzingcomprises visualizing the chlorotoxin conjugate optically. 370. Themethod of embodiment 367, wherein the imaging, visualizing, or analyzingcomprises in vivo, or ex vivo imaging, visualizing, or analyzing. 371.The method of embodiment 370, wherein the imaging, visualizing, oranalyzing comprises optically imaging the sarcoma. 372. The method ofembodiment 367, wherein the chlorotoxin conjugate comprises one or morelabeling agents. 373. The method of embodiment 372, wherein the labelingagent comprises a fluorescent moiety. 374. The method of embodiment 373,wherein the fluorescent moiety comprises a near infrared fluorescentmoiety. 375. The method of embodiment 372, wherein the labeling agentcomprises a radionuclide. 376. The method of embodiment 367, wherein thesoft-tissue sarcoma is in subcutaneous fatty tissue. 377. The method ofembodiment 367, wherein the detecting is performed during or related tosurgery or resection. 378. A method of detecting and removingsoft-tissue sarcoma in an individual comprising the steps of: c)administering a chlorotoxin conjugate to the individual, wherein thechlorotoxin conjugate binds to the squamous cell carcinoma or cutaneouscell carcinoma; and d) imaging, visualizing, or analyzing the boundchlorotoxin conjugate, and removing the tissue bound by the chlorotoxinconjugate. 379. A method of detecting cutaneous squamous cell carcinomain an individual, comprising the steps of: e) administering achlorotoxin conjugate to the individual, wherein the chlorotoxinconjugate binds to the cutaneous squamous cell carcinoma; and f)imaging, visualizing, or analyzing the bound chlorotoxin conjugate. 380.The method of embodiment 379, wherein the detecting comprises in vivo,or ex vivo detection. 381. The method of embodiment 379, wherein theimaging, visualizing, or analyzing comprises visualizing the chlorotoxinconjugate optically. 382. The method of embodiment 381, wherein theimaging, visualizing, or analyzing comprises in vivo, or ex vivoimaging, visualizing, or analyzing. 383. The method of embodiment 382,wherein the imaging, visualizing, or analysis comprises opticallyimaging the cutaneous squamous cell carcinoma. 384. The method ofembodiment 379, wherein the chlorotoxin conjugate comprises one or morelabeling agents. 385. The method of embodiment 384, wherein the labelingagent comprises a fluorescent moiety. 386. The method of embodiment 385,wherein the fluorescent moiety comprises a near infrared fluorescentmoiety. 387. The method of embodiment 384, wherein the labeling agentcomprises a radionuclide. 388. A method of detecting and removingcutaneous squamous cell carcinoma in an individual comprising the stepsof: g) administering a chlorotoxin conjugate to the individual, whereinthe chlorotoxin conjugate binds to the cutaneous squamous cellcarcinoma; and h) imaging, visualizing, or analyzing the boundchlorotoxin conjugate, and removing the tissue bound by the chlorotoxinconjugate. 389. The method of embodiment 388, wherein the cutaneoussquamous cell carcinoma, or a portion thereof, is removed during orrelated to surgery. 390. A method of detecting a low-grade tumor in anindividual, comprising the steps of: i) administering a chlorotoxinconjugate to the individual, wherein the chlorotoxin conjugate binds tothe low-grade tumor; and j) imaging, visualizing, or analyzing the boundchlorotoxin conjugate. 391. The method of embodiment 390, wherein thedetecting comprises in vivo, or ex vivo detection. 392. The method ofembodiment 390, wherein the imaging, visualizing, or analyzing comprisesvisualizing the chlorotoxin conjugate optically. 393. The method ofembodiment 392, wherein the imaging, visualizing, or analyzing comprisesin vivo, or ex vivo imaging, visualizing, or analyzing. 394. The methodof embodiment 393, wherein the imaging, visualizing, or analyzingcomprises optically imaging the low-grade tumor. 395. The method ofembodiment 390, wherein the chlorotoxin conjugate comprises one or morelabeling agents. 396. The method of embodiment 395, wherein the labelingagent comprises a fluorescent moiety. 397. The method of embodiment 396,wherein the fluorescent moiety comprises a near infrared fluorescentmoiety. 398. The method of embodiment 395, wherein the labeling agentcomprises a radionuclide. 399. The method of embodiment 390, wherein thedetecting is performed during or related to surgery or resection. 400. Amethod of detecting and removing a low-grade tumor in an individualcomprising the steps of: k) administering a chlorotoxin conjugate to theindividual, wherein the chlorotoxin conjugate binds to the low-gradetumor; and 1) imaging, visualizing, or analyzing the bound chlorotoxinconjugate, and removing the tissue bound by the chlorotoxin conjugate.401. The method of embodiment 390, wherein the low-grade tumor selectedfrom the group consisting of: m) a low-grade tumor in or from braintissue; n) a low-grade tumor in or from subcutaneous fatty tissue; ando) a low-grade tumor in or from breast or mammary tissue. 402. A methodfor detecting a tumor in an individual comprising the steps of: p)administering a chlorotoxin conjugate to the individual wherein thechlorotoxin conjugate binds to the tumor, and wherein the chlorotoxinconjugate is administered in an amount of between about 3 mg to about 6mg; and q) imaging, visualizing, or analyzing the bound chlorotoxinconjugate. 403. The method of embodiment 402, wherein the detectingcomprises in vivo, or ex vivo detection. 404. The method of embodiment403, wherein the imaging, visualizing, or analyzing comprisesvisualizing the chlorotoxin conjugate optically. 405. The method ofembodiment 404, wherein the imaging, visualizing, or analyzing comprisesin vivo, or ex vivo imaging, visualizing, or analyzing. 406. The methodof embodiment 404, wherein the imaging, visualizing, or analyzingcomprises optically imaging the tumor. 407. The method of embodiment402, wherein the chlorotoxin conjugate comprises one or more labelingagents. 408. The method of embodiment 407, wherein the labeling agentcomprises a fluorescent moiety. 409. The method of embodiment 408,wherein the fluorescent moiety comprises a near infrared fluorescentmoiety. 410. The method of embodiment 407, wherein the labeling agentcomprises a radionuclide. 411. The method of embodiment 402, wherein thedetecting is performed during or related to surgery or resection. 412.The method of embodiment 411, wherein the tumor, or a portion thereof,is removed during or related to surgery. 413. A method of detecting andremoving a tumor in an individual comprising the steps of: r)administering a chlorotoxin conjugate to the individual, wherein thechlorotoxin conjugate binds to the tumor, and wherein the chlorotoxinconjugate is administered in an amount of between about 0.9 mg/m² toabout 1.1 mg/m²; s) imaging, visualizing, or analyzing the boundchlorotoxin conjugate, and t) removing the tissue bound by thechlorotoxin conjugate. 414. A method of detecting and removing a tumorin an individual comprising the steps of: u) administering a chlorotoxinconjugate to the individual, wherein the chlorotoxin conjugate binds tothe tumor, and wherein the chlorotoxin conjugate is administered in anamount of between about 3 mg to about 6 mg; v) imaging, visualizing, oranalyzing the bound chlorotoxin conjugate, and w) removing the tissuebound by the chlorotoxin conjugate. 415. The method of any of thepreceding embodiments, wherein the chlorotoxin conjugate comprises achlorotoxin and a detectable label. 416. The method of embodiment 415,wherein the chlorotoxin comprises a sequence having at least 85%sequence identity to the sequence ofMCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X is selected from K, Aand R. 417. The method of embodiment 415 or 416, wherein the detectablelabel comprises a fluorescent moiety. 418. The method of embodiment 417,wherein the fluorescent moiety comprises a near infrared fluorescentmoiety. 419. The method of embodiment 415, wherein the detectable labelcomprises a radionuclide. 420. The method of embodiment 415, wherein thedetectable label comprises a near-infrared dye. 421. The method ofembodiment 415, wherein the detectable label comprises a cyanine dye.422. The peptide of embodiment 420, wherein the near-infrared dyeselected from the group consisting of Cy5.5, DyLight 750, indocyaninegreen (ICG) and IRdye 800. 423. The method of any one of the precedingembodiments, wherein the detecting comprises in vivo, or ex vivodetection. 424. The method of any one of embodiments 367-423, whereinthe imaging, visualizing, or analyzing the bound chlorotoxin conjugateis performed on a sample. 425. The method of any one of embodiments367-424, wherein the imaging, visualizing, or analyzing comprisesvisualizing the chlorotoxin conjugate optically. 426. The method of anyone of embodiments 367-425, wherein the imaging, visualizing, oranalyzing comprises optically imaging the tumor. 427. The method ofembodiment 426, wherein the tumor is a sarcoma. 428. The method ofembodiment 427, wherein the sarcoma is a soft-tissue sarcoma. 429. Themethod of any one of embodiments 367-428, wherein the chlorotoxinconjugate comprises one or more labeling agents. 430. The method ofembodiment 428, wherein the soft-tissue sarcoma is in subcutaneous fattytissue. 431. The method of any one of embodiments 367-430, wherein thedetecting is performed during or related to surgery or resection. 432.The method of any one of embodiments 367-431, wherein the tumor can bedifferentiated from adjacent or uninvolved tissue with about 90%sensitivity and about 90% specificity. 433. The method of any one ofembodiments 367-432, wherein the tumor can be differentiated fromadjacent or uninvolved tissue with at least 94% sensitivity and about100% specificity. 434. The method of embodiment 432 or 433, wherein thetumor is a sarcoma. 435. The method of embodiment 434, wherein thesarcoma is a soft-tissue sarcoma. 436. The method of any one ofembodiments 367-435, wherein the tumor selected from the groupconsisting of: x) a tumor in or from brain tissue; y) a tumor in or fromsubcutaneous fatty tissue; and a tumor in or from breast or mammarytissue. 437. The method of embodiment 436, wherein the tumor of a), b)or c) is a low-grade tumor. 438. The method of any one of embodiments367-437, wherein the chlorotoxin conjugate comprises a chlorotoxin and adetectable label. 439. The method of embodiment 438, wherein thechlorotoxin comprises a sequence having at least 85% sequence identityto the sequence of MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR, wherein X isselected from K, A and R. 440. The method of embodiment 438, wherein thedetectable label comprises a fluorescent moiety. 441. The method ofembodiment 440, wherein the fluorescent moiety comprises a near infraredfluorescent moiety. 442. The method of embodiment 438, wherein thedetectable label comprises a radionuclide. 443. The method of embodiment438, wherein the detectable label comprises a near-infrared dye. 444.The method of embodiment 438, wherein the detectable label comprises acyanine dye. 445. The peptide of embodiment 443, wherein thenear-infrared dye selected from the group consisting of Cy5.5, DyLight750, indocyanine green (ICG) and IRdye 800. 446. The method of any oneof embodiments 367-445, wherein the detecting comprises in vivo, or exvivo detection. 447. The method of any one of embodiments 367-446,wherein the imaging, visualizing, or analyzing the bound chlorotoxinconjugate is performed on a sample. 448. The method of any one ofembodiments 367-447, wherein the imaging, visualizing, or analyzingcomprises visualizing the chlorotoxin conjugate optically. 449. Themethod of any one of embodiments 367-448, wherein the chlorotoxinconjugate comprises one or more labeling agents. 450. The method any oneof embodiments 367-449, wherein the detecting is performed during orrelated to surgery or resection. 451. A method of detecting soft-tissuesarcoma in an individual comprising: administering a chlorotoxinconjugate to the individual, wherein the chlorotoxin conjugate comprisesa detectable agent and a chlorotoxin polypeptide having at least 85%sequence identity with MCMPCFTTDHQMARXCDDCCGGXGRGXCYGPQCLCR; binding thechlorotoxin conjugate to the soft-tissue sarcoma; and detecting thebound chlorotoxin conjugate, wherein an elevated level of boundchlorotoxin conjugate indicates the presence of soft-tissue sarcoma.452. The method of embodiment 367, wherein the administration of thechlorotoxin conjugate prevents the spread of or metastasis of the tumor.453. The method of embodiment 368, wherein the chlorotoxin conjugatecomprises a chemotherapeutic, an anti-cancer agent, or an anti-cancerdrug. 454. The method of embodiment 367, wherein the chlorotoxinconjugate is administered in conjunction with detecting a central orprimary tumor. 455. The method of embodiment 371, wherein theadministration of the chlorotoxin conjugate prevents the spread of ormetastasis of the tumor. 456. The method of embodiment 372, wherein thechlorotoxin conjugate comprises a chemotherapeutic, an anti-canceragent, or an anti-cancer drug. 457. The method of embodiment 371,wherein the chlorotoxin conjugate is administered in conjunction withdetecting a central or primary tumor. 458. The method of embodiment 376,wherein the administration of the chlorotoxin conjugate prevents thespread of or metastasis of the tumor. 459. The method of embodiment 376,wherein the chlorotoxin conjugate comprises a chemotherapeutic, ananti-cancer agent, or an anti-cancer drug. 460. The method of embodiment376, wherein the chlorotoxin conjugate is administered in conjunctionwith detecting a central or primary tumor.

EXAMPLES

The invention is further illustrated by the following non-limitingexamples.

Example 1

Stability of Compound 16 with Ammonium Acetate Salt

This example demonstrates the production and stability of Compound 16under various pH and temperature storage conditions over time.

The peptide portion of Compound 16 is a targeting peptide (modifiedchlorotoxin) conjugated to the fluorescent dye. The targeting peptidebinds selectively to cancerous cells and the dye portion facilitatesdetection via imaging. The peptide is a 36 amino acid modifiedchlorotoxin (having a sequence ofH-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH)wherein two of three lysine amino acids in native chlorotoxin aresubstituted with arginine (K15R and K23R) to facilitate the subsequentconjugation with a fluorophore to the single remaining lysine (K27)residue resulting in a mono-labeled fluorescent active pharmaceuticalingredient.

Methods and Results: Product solutions were stored at <8° C. duringmanufacturing. All analysis utilized the in-process HPLC method PR todetermine the stability of the product in the different buffers.

Dye Conjugation: 500 mg of the peptide portion of Compound 16:(TFAsalt), (net peptide content 76.7, net peptide 384 mg, 0.095 mmo1) weredissolved in a solution of sodium bicarbonate. DMSO and 140 mg ofICG-Sulfo-ATT dissolved in dried DMSO (net dye 126 mg 0.152 mmo1, 1.6eq. based on a product activity of 90%) were added, resulting in a finalreaction volume of approximately 200 mL. The reaction was followed byRP-HPLC and considered completed after 3 hours, with ˜3.4% un-reactedCompound 16 remaining.

Ammonium Bicarbonate Purification: The reaction solution was filteredand then diluted with 400 mL of water. The solution was loaded on anRP-HPLC column equilibrated with 0.1M ammonium bicarbonate, and theproduct recovered by applying a linear gradient of acetonitrile.

The product eluted as a single peak. Fractions were collected andanalyzed by analytical HPLC. During analysis the fractions were storedat ≤8° C. protected from light.

The purity, estimated concentration, and estimated amount of product ineach fraction are reported in Table 15. The estimation was based on thepeak area observed during release analysis of a solution of the productat a concentration of 1 mg/mL in water.

TABLE 15 Ammonium Bicarbonate Purification Part I Concentration FractionNumber Purity (%) (mg/mL) Amount (mg) 6-1-3 5.48 0.0025 0.1 6-1-4 73.360.0970 4.8 6-1-5 98.67 1.2638 63.2 6-1-6 98.96 3.0056 150.3 6-1-7 95.592.0464 102.3 6-1-8 92.16 1.0622 53.1 6-1-9 81.87 0.3959 19.8 6-1-1066.56 0.0703 3.5 Total product recovery in fractions 3 to 10 303 Productrecovery in main pool (fractions 5 to 8) 282

The main pool (fractions 6-1-5 to 6-1-8, purity 97%) was combined andtransferred to the salt exchange step. Samples of the main pool wereanalyzed for a period of 5 day of storage protected from light at ≤8° C.The material was stable under these conditions.

Ammonium Acetate Salt Exchange: The primary purification main poolsolution (˜200 mL) was diluted with 100 mL of water and loaded on anRP-HPLC column equilibrated with 0.1M ammonium bicarbonate. Followingloading of the sample, the column was equilibrated with 4 bed volumes of0.1M ammonium acetate adjusted to pH 7.6 with ammonium hydroxide.Finally the column was equilibrated with 0.01M ammonium acetate pH 7.6and the product was recovered by applying a linear gradient of 0.01Mammonium acetate pH 7.6 in 75% Acetonitrile.

The product eluted as a single peak at approximately 39% acetonitrileconcentration. The fractions were collected and analyzed by analyticalHPLC. During analysis, the fractions were stored at <8° C. protectedfrom light.

The purity, estimated concentration, and estimated amount of product ineach fraction are reported in Table 16. The estimation was based on thepeak area observed during release analysis of a solution of the productat a concentration of 1 mg/mL in water.

TABLE 16 Ammonium Bicarbonate Purification Part II ConcentrationFraction Number Purity (%) (mg/mL) Amount (mg) 7-1-1 79.1 0.020084 1.07-1-2 98.6 1.115285 55.8 7-1-3 99.0 1.804414 90.2 7-1-4 98.1 1.19846459.9 7-1-5 94.8 0.425017 21.3 7-1-6 84.2 0.101113 5.1 7-1-7 65.40.037211 1.9 7-1-8 55.5 0.021173 1.1 Total product recovery in fractions3 to 10 236 Product recovery in main pool (fractions 5 to 8) 232

The main pool (fractions 7-1-2 to 7-1-6, purity 97.2%) were combined andtransferred to lyophilization. Samples of the main pool were analyzedfor a period of 5 day of storage protected from light at ≤8° C. Thematerial was stable under these conditions. Dilution with a volume equalto approximately half the main pool volume provided a stable solutionwithout the presence of a precipitate. This dilution was utilized at thepreparative scale.

Lyophilization: The main pool at stage 7 was diluted with 100 mL ofwater and lyophilized over a period of 4 days using a bottlelyophilizer. There was no problem with solubility after dilution as hadbeen observed with the ammonium bicarbonate main pool; a sample wasdiluted to 15% acetonitrile without any observed precipitation. Duringlyophilization, the product formed a stable self-supporting cake.

Reconstitution: The product was readily soluble in water at 1, 5 and 10mg/mL. Water was selected as the reconstitution solution. Additionally,an LC-MS analysis was performed on the final material.

A sample of the main pool of Stage 6 (ammonium bicarbonate purification)was stored without dilution at <8° C. for 5 days protected from light.The sample was analyzed daily. Results are reported in Table 17. Theresults indicate a low level of instability.

TABLE 17 Stage 6 Main Pool Stability Time point (hour) Purity (%) 0 96.626 96.4 48 96.4 72 96.3 105 95.7 191 95.4

A sample of the main pool of Stage 7 (ammonium acetate purification) wasstored without dilution at <8° C. for 5 days protected from light. Thesample was analyzed daily. Results are reported in Table 18. The resultsindicate a low level of instability.

TABLE 18 Stage 7 Main Pool Stability Time point (hour) Purity (%) Zero97.8 26 97.6 48 97.4 79 97.1 91 96.8 610 96.4

An optimized and scalable conjugation procedure for production ofCompound 16 for GMP manufacturing was developed. Two different dyereagents were evaluated: ICG-Sulfo-NHS ester and ICG-Sulfo-ATT (bothresulting in Compound 16). Design of Experiments (DOE) studies wasprepared.

After conjugation of Compound 16, the crude peptide conjugate waspurified by RP-HPLC with a TFA and acetonitrile gradient elution.Fractions were characterized for purity with a RP-HPLC assay, pooled anddesalted into acetate by HPLC prior to lyophilization to bulk conjugateproduct.

Compound 16 was stable for up to 14 days at pH 7.5 and 8.5 when storedin the dark at 4° C. in 10 mM Tris, 5% Dextrose and is sensitive to lowpH and temperatures above 4° C.

Example 2 Evaluation of the Stability of Compound 16 in Various Buffersand at Various pHs

This example shows the stability of Compound 16 with respect to pH andprovides an evaluation of the use of alternative buffers and variousexcipients and configurations.

This example further shows that various classes of excipients protectCompound 16 from thermal, photo, oxidative and freeze/thaw stress.

In some cases, formulations were stored in microcentrifuge tubes with anair atmosphere and assayed after 2d and 5d at 40° C. in the dark, after3d at room remperature (rt) in the dark (dk) or in ambient light (lt),and after 3X freeze thaw (F/T) between −20° C. and room temperature inthe dark. Formulations were evaluated by visual examination,centrifugation, RP-HPLC, concentration by A⁷⁸⁶, pH, and SDS-PAGE.

In some cases, the timepoints were 4, 7, and 14 days at room temperaturewith evaluation by visual examination, centrifugation, RP-HPLC,concentration by A⁷⁸⁶, pH, and SDS-PAGE. Where extra material remained,additional stressing conditions were evaluated for further information.The formulations were contained in microcentrifugation tubes in an airenvironment.

Methods: The following stock solutions were prepared and passed througha 0.2 μm filter (Table 19). Dialysis buffers were prepared immediatelybefore use by combining a stock from Table 19 with sparged water(sparged by bubbling with argon for 15 min-3 h).

TABLE 19 Stock Buffer Solutions Buffer Molarity pH Tris 0.5 7.0, 7.5,8.0, 8.5 His 0.25 5.5, 6.5, 7.0 maleic acid 0.5 7.0 HEPES 0.5 7.0 EDA0.5 7.0 acetic 0.5 4.5

Compound 16 was prepared at 3 mg/mL in water (see concentration by A⁷⁸⁶below). Slide-a-lyzer cassettes were pre-soaked in water, then theCompound 16 was dispensed into the cassettes (˜11 mL for formulation 1,˜5 mL for formulations 2-6, ˜2.7 mL for formulations 7-12). Thecassettes were placed into beakers containing 550 mL of thecorresponding formulation buffer. Samples were formulated by dialyzingwith stirring in the dark at room temperature (on a multiposition stirplate, covered with a box covered with aluminum foil). Dialysis bufferwas changed after 1 h, again after 1.25 h, and allowed to continueovernight.

For some formulations, the formulation was placed in a fresh tube andcombined with additional sterile 0.25M or 0.5M buffer stock to yield anew formulation with 30 mM concentration of buffer. All formulationswere then dispensed into 1.5 mL microcentrifuge tubes, 0.5 mL per tube.

The designed stability timepoints were 4, 7, and 14 days at roomtemperature. Where extra material remained, additional testing wasperformed. One tube of each formulation was assayed immediately (“t0”).For additional information, remaining material from these t0 tubes wasthen frozen at −20° C., then thawed at 5° C., and assayed for pH andconcentration (“1× F/T”). Other tubes were incubated in the dark at roomtemperature (approximately 22-23° C.). A tube was removed and assayed at4, 7, and 14d room temperature (rt) (“4d rt”, “7d rt”, “14d rt”). Foradditional information, after the 7d room temperature sample wasassayed, the remaining material was placed at room temperature in thelight for one day and then assayed again (“7+1d light”). These tubeswere placed on their sides on the benchtop. Stability samples wereassayed by visual inspection, centrifugation, RP-HPLC, A₇₈₆, pH, and, insome cases, SDS-PAGE.

Samples were examined visually for clarity, color, and visibleparticulates. They were then mixed, withdrawn into glass Pasteur pipets,and examined again. Samples were centrifuged ˜10,000 rpm for ˜2 min.Samples were then examined visually for any visible pellet.

Samples were analyzed by RP-HPLC using the Zorbax_10 method.

TABLE 20 Tested Formulations # Composition pH 1, (1B) 10, (30) mM Tris7.0 2, (2B) 10, (30) mM His 7.0 3 10 mM maleic acid 7.0 4, (4B) 10, (30)mM EDA 7.0 6, (6B) 10, (30) mM HEPES 7.0 7 10 mM acetic 4.5 8 10 mM His5.5 9 10 mM His 6.5 10 10 mM Tris 7.5 11 10 mM Tris 8.0 12 10 mM Tris8.5

100 μl of sample was placed in an HPLC vial insert, placed in an amberHPLC vial, and held at 2-8° C. before the 2 μl injection. Theformulation buffer was injected twice at the beginning of each run, anda matched formulation buffer blank was run each time before injection ofa sample in a new buffer.

The spectrophotometer was blanked with matching formulation buffer.Concentration was calculated using the extinction coefficient. The pHwas measured using a calibrated micro pH electrode.

Samples were prepared per the manufacturer's recommendations and thenheated at 70° C. for 10 min. 2 μg in 10 μL was loaded onto the SDS-PAGEgels and then run at 200V for approximately 35 min. Afterelectrophoresis, the gels were washed in water for 5 min at roomtemperature, 3 times. Then they were stained at room temperature for 1.5h, destained in water overnight, and then destained again with water andimaged on the same day.

Results. Formulations were produced by dialyzing a stock of Compound 16at 3 mg/mL in water into various buffers. The formulations were thendispensed into microcentrifuge tubes, in air, and stored in the dark atroom temperature for up to 14 days. The results from visual examinationof the formulations are given in Table 21.

TABLE 21 Visual Days at rt Formulation 7 + 1 d # Composition pH 0 4 7 14light 1 10 mM tris 7.0 CGN CGN CGN CGN CGN 2 10 mM His 7.0 CGN CGN CGFCGN CGF 2B 30 mM His 7.0 CGF CGF CGF CGN nt 3 10 mM maleic acid 7.0insoluble nt nt nt nt 4 10 mM EDA 7.0 CGN CGF CGN CGF CGN 6 10 mM HEPES7.0 CGF CGN CGN CGN CGN 6B 30 mM HEPES 7.0 CGN CGF CGF CGN Nt 7 10 mMacetic 4.5 CGN CGF CGN CGN CGN 8 10 mM His 5.5 CGN CGN CGN CGN CGN 9 10mM His 6.5 CGF CGN CGN CGN CGN 10 10 mM tris 7.5 CGF CGF CGN CGF nt 1110 mM tris 8.0 CGN CGN CGN CGN CGF 12 10 mM tris 8.5 CGF CGF CGF CGF CGFVisual Inspection. C = clear; G = emerald green; N = essentially novisible particles; F = very few particles or fibers visible; nt = nottested.

The results after centrifugation are provided in Table 22. The pHmeasurements are given in Table 23.

TABLE 22 Pellet Analysis after Centrifugation. CentrifugationFormulation Days at rt # Composition pH 7 14 7 + 1 d light 1 10 mM tris7.0 N N N 2 10 mM His 7.0 N N N 4 10 mM EDA 7.0 N N N 6 10 mM HEPES 7.0N N N  6B 30 MM HEPES 7.0 N N nt 7 10 mM acetic 4.5 N N N 8 10 mM His5.5 N N N 9 10 mM His 6.5 N N N 10  10 mM tris 7.5 P P nt 11  10 mM tris8.0 P P P 12  10 mM tris 8.5 P P P N = no pellet; P = pellet.

TABLE 23 pH stability analysis. pH Formulation Days at rt F/T #Composition pH 4 7 14 1X F/T Buffer alone 1 10 mM tris 7.0 7.0 6.8 6.86.9 7.0 2 10 mM His 7.0 6.9 6.8 6.9 6.8 6.9   2B 30 mM His 7.0 6.9 6.86.9 6.8 nt 4 10 mM EDA 7.0 6.6 6.6 6.6 6.6 6.7 6 10 mM HEPES 7.0 7.0 6.97.0 7.0 6.9   6B 30 MM HEPES 7.0 7.0 6.9 6.9 6.9 nt 7 10 mM acetic 4.54.6 4.6 4.6 4.6 4.5 8 10 mM His 5.5 5.4 5.3 5.4 5.3 5.4 9 10 mM His 6.56.4 6.4 6.4 6.4 6.4 10  10 mM tris 7.5 7.4 7.3 7.4 7.4 7.4 11  10 mMtris 8.0 7.9 7.8 7.9 7.8 7.9 12  10 mM tris 8.5 8.3 8.3 8.3 8.3 8.4

The % main peak by RP-H PLC is given in Table 24. Stability drops offrapidly at pH ≤6.5.

TABLE 24 % Main Peak by RP-HPLC; % Main peak Formulation Days at rt #Composition pH 0 4 7 14 1 10 mM tris 7.0 97.9% 96.8% 95.5% 93.5% 2 10 mMHis 7.0 98.0% 96.1% 94.8% 90.3%   2B 30 mM His 7.0 97.7% 96.2% 94.7%91.9% 4 10 mM EDA 7.0 98.3% 97.1% 96.2% 94.6% 6 10 mM HEPES 7.0 97.0%96.6% 94.2% 91.0%   6B 30 mM HEPES 7.0 97.6% 96.2% 94.9% 92.6% 7 10 mMacetic 4.5 92.6% 64.8% 47.4% 21.6% 8 10 mM His 5.5 97.1% 90.9% 87.1%75.8% 9 10 mM His 6.5 97.8% 95.7% 94.2% 90.9% 10  10 mM tris 7.5 97.7%96.7% 96.0% 94.0% 11  10 mM tris 8.0 97.7% 97.2% 96.6% 93.4% 12  10 mMtris 8.5 97.9% 95.5% 93.9% 91.7% Δ light v dark = (7 + 1 d light) − (7 ddark).

FIG. 1A-FIG. 1C shows SDS-PAGE analysis of the formulations after 14days at room temperature. FIGS. lA and 1B show SDS-PAGE analysis of,from left to right, (molecular weight marker) MWM, 1, 2, 2B, 4, 6, 6B,9, 10 and reference. FIG. 1A was performed with a reducing agent andFIG. 1B was performed without a reducing agent. FIG. 1C shows SDS-PAGEanalysis of, from left to right, 7, 8, 11, 12, reference and MWM (noreducing agent). (Reference=Pilot Lot, Sublot #2. MWM: 188k, 98k, 62k,49k, 38k, 28k, 17k, 14k, 6k, 3k. Arrow points to a higher molecularweight species.

Compound 16 was formulated by additional means. Stocks were prepared andthe pH of some stocks was adjusted to near 6.8 to avoid pH shift uponaddition to formulations (Table 25). All stocks were 0.2 μm filteredexcept BHT, BHA, propyl gallate, and polysorbates. Polysorbate and Nalstocks were stored with an argon blanket. His, Met, Nal, polysorbate,BHT, BHA, and propyl gallate stocks were stored in the dark.

TABLE 25 Stock Buffer Solutions Component mM pH Tris 500 7.5 His 250 7.5NaCl 4000 Met 200 ~6.8 EDTA 200 ~6.8 Gly 200 ~6.8 Component w/v %Solvent Mannitol 10% H₂O Sucrose 20% H₂O Trehalose*2H2O 22% H₂O BHT2.50% 10% H2O in EtOH BHA 2.50% 50% EtOH/H₂O Propyl gallate 2.50% 25%EtOH in H₂O PS80 10% H₂O PS20 10% H₂O Nal 20% H₂O HPCD 40% H₂O Captisol40% H₂O

Compound 16 was prepared at 10 mg/mL in argon-sparged water. TheCompound 16 stock was combined with buffer stock, osmolyte stock, andwater to make parent stocks A-E containing Compound 16 (Table 26). ThepH of each soluble parent stock was adjusted (from starting values of6.6-6.7) to 6.8 with 0.1N NaOH.

TABLE 26 Parent Stock Formulations mLs: Parent Stock composition Cmpd 16Parent Stocks mM Buffer Osmolyte stock Total mL Buffer Buffer Osmolyte[Osmolyte] stock stock (10 mg/mL) H2O A 4.50 Tris 11.7 mannitol 5.8%0.105 2.625 1.575 0.195 B 24.15 His 11.7 mannitol 5.8% 1.127 14.0888.453 0.483 C 2.4 His 11.7 sucrose 11.1% 0.112 1.330 0.840 0.118 D 2.4His 11.7 trehalose 12.3% 0.112 1.336 0.840 0.112 E 0.1 His 11.7 NaCL(mM) 163.3 0.005 0.004 0.035 0.056

The final formulations were then created by combining the parent stockswith other excipient stocks and water. The formulations were passedthrough 0.2 pm sterile syringe filters. 0.4 mL of Formulations 1, 3, 4,and 5 were reserved for “t0” analysis.

Two 0.3 mL tubes were placed in a dark 40° C_(max) incubator foranalysis after 2d and 5d. The 2d samples were assayed after 40 h (1.7d).One 0.3 mL tube was subjected to “3X F/T.” 1X F/T comprised freezing thematerial at −20° C. for at least 8h, thawing the material at roomtemperature (rt) in the dark (˜1 h), then gently mixing, inverting, andspinning to bring the material back to the bottom of the tube. Thiscycle was performed a total of three times. The remaining 0.3 mL tubewas reserved at 5° C. One of the 0.5 mL tubes was placed in the dark atroom temperature and the other 0.5 mL tube was placed in the light atroom temperature. For light exposure, the tubes were placed on theirside and exposed to ambient light. A volume of 0.5 mL was chosen forlight exposure, as previous work had shown 0.3 mL samples are prone toprecipitation and degradation after light exposure in thisconfiguration.

Stability samples were assayed by visual inspection, centrifugation,RP-HPLC, A₇₈₆, pH, and, in some cases, SDS-PAGE. Samples were held inthe dark at 5° C. when not in use. Samples were examined visually forclarity, color, and visible particulates. Samples were inverted threetimes and examined again. Samples were gently mixed, then centrifuged10,000 rpm for ˜2 min. Samples were examined visually for a pellet.Remaining assays were performed on the supernatant.

Samples were prepared per the manufacturer's recommendations and thenheated at 70° C. for 10 min. 2.5 μg in 10 μL was loaded onto theSDS-PAGE gels and then run at 165-200V. After electrophoresis, the gelswere washed in water for 5 min at room temperature, 3 times. Gels werestained at room temperature for lh, destained in water overnight, andthen destained again with water and imaged on the same day.

Formulations were produced by dissolving Compound 16 at 10 mg/mL inwater, diluting into the buffer and osmolyte, adjusting the pH, addingfinal additives and water as needed (Table 27).

TABLE 27 Formulations Tested at 3 mg/mL Compound 16 at pH 6.8 # BufferOsmolyte [Osm] Other [Other] Parameter examined 1 10 mM Tris mannitol 5%— Buffer 3 10 mM His mannitol 5% — Buffer 4 10 mM His sucrose 9.5%  —Osmolyte 5 10 mM His trehalose 10.5%   — Osmolyte 6 10 mM His NaCl 140mM — Ionic strength 7 10 mM His mannitol 5% NaCl 10 mM Ionic strength 810 mM His mannitol 5% Met 10 mM Antioxidant 9 10 mM His mannitol 5% BHT0.01% Antioxidant 10 10 mM His mannitol 5% BHA 0.01% Antioxidant 11 10mM His mannitol 5% propyl gallate 0.01% Antioxidant 12 10 mM Hismannitol 5% EDTA  1 mM Antioxidant synergist (metal chelator) 15 10 mMHis mannitol 5% Gly 20 mM Amino acid 16 10 mM His mannitol 5% PS80 0.02%Surfactant 17 10 mM His mannitol 5% PS20 0.02% Surfactant 19 10 mM Hismannitol 5% HPCD   5% Cyclodextrin

Formulations #6 (140 mM NaCI), #12 (1 mM EDTA) and #18 (0.2% Nal) hadimmediate gross precipitation and were discarded. In all otherformulations, the material was clear, green, and had no significantvisible particle formation at all timepoints tested (Table 28).

TABLE 28 Visual Inspection. Visual Formulation 2 d 5 d 3 d 3 d 3X #Buffer Osmolyte Other [Other] t0 40 C. 40 C. rt dk rt lt FT 1 Trismannitol — CGF CGN CGF CGN CGN CGN 3 His mannitol — CGN CGN CGN CGN CGNCGN 4 His sucrose — CGN CGN CGN CGN CGN CGN 5 His trehalose — CGN CGNCGN CGN CGN CGN 6 His 140 mM — insoluble nt nt nt nt nt NaCl 7 Hismannitol NaCl 10 mM nt CGN nt nt nt CGN 8 His mannitol Met 10 mM nt CGNCGN CGN CGN CGN 9 His mannitol BHT 0.01% nt CGN CGN CGN CGN CGN 10 Hismannitol BHA 0.01% nt CGN CGN CGN CGN CGN 11 His mannitol propyl 0.01%nt CGN CGN CGN CGN CGN gallate 12 His mannitol EDTA  1 mM insoluble ntnt nt nt nt 15 His mannitol Gly 20 mM nt CGN CGN CGF CGN CGN 16 Hismannitol PS80 0.02% nt CGN CGN CGF CGN CGN 17 His mannitol PS20 0.02% ntCGN CGN CGN CGN CGN 19 His mannitol HPCD   5% nt CGN CGN CGN CGN CGN C =clear; G = emerald green; N = essentially no visible particles; F = veryfew particles or fibers visible; P = pellet, nt = not tested.

All samples were at pH 6.8 +/−0.1 (Table 29).

TABLE 29 pH pH Formulation 2 d 5 d 3 d 3 d 3X # Buffer Osmolyte Other[Other] t0 40 C. 40 C. rt dk rt lt FT 1 Tris mannitol — 6.8 6.8 6.8 6.96.8 6.9 3 His mannitol — 6.8 6.8 6.8 6.9 6.8 6.8 4 His sucrose — 6.8 6.86.8 6.9 6.8 6.9 5 His trehalose — 6.8 6.8 6.8 6.8 6.8 6.8 7 His mannitolNaCl 10 mM nt 6.8 nt nt nt 6.8 8 His mannitol Met 10 mM nt 6.8 6.8 6.86.8 6.8 9 His mannitol BHT 0.01% nt 6.8 6.8 6.8 6.8 6.8 10 His mannitolBHA 0.01% nt 6.8 6.8 6.8 6.8 6.8 11 His mannitol propyl gallate 0.01% nt6.8 6.7 6.9 6.8 6.8 15 His mannitol Gly 20 mM nt 6.8 6.8 6.8 6.8 6.8 16His mannitol PS80 0.02% nt 6.8 6.8 6.9 6.8 6.8 17 His mannitol PS200.02% nt 6.8 6.8 6.8 6.8 6.8 19 His mannitol HPCD   5% nt 6.8 6.8 6.86.8 6.8

The % main peak by RP-HPLC is given in Table 30.

TABLE 30 % Main Peak by RP-HPLC. % Main Peak Formulation 2 d 5 d 3 d 3X# Buffer Osmolyte Other [Other] t0 40 C. 40 C. rt lt FT 1 Tris mannitol— 97.5% 93.9% 85.1% 73.9% 97.4% 3 His mannitol — 98.5% 94.0% 86.1% 92.6%98.6% 7 His mannitol NaCl 10 mM nt 95.3% nt nt 98.3% 8 His mannitol Met10 mM nt 94.3% 87.5% 93.0% 98.6% 9 His mannitol BHT 0.01% nt 94.1% 83.4%91.2% 98.7% 10 His mannitol BHA 0.01% nt 93.9% 84.9% 91.1% 98.3% 11 Hismannitol propyl 0.01% nt 88.7% 72.7% 89.7% 98.2% gallate 15 His mannitolGly 20 mM nt 94.4% 83.9% 90.9% 97.7% 16 His mannitol PS980 0.02% nt93.4% 83.8% 91.9% 98.6% 17 His mannitol PS20 0.02% nt 94.4% 84.3% 90.3%98.3% 19 His mannitol HPCD   5% nt 87.3% 65.6% 89.3% 96.2% Δ light vs.dark = % main peak after 3 d room temperature in the light minus % mainpeak after 3 d room temperature in the dark.

After 5 days at 40° C., formation of significant amounts of highermolecular weight species (HMWS) were visible by reducing and nonreducingSDS-PAGE (FIG. 2A-FIG. 2C). FIG. 2A-FIG. 2C shows SDS-PAGE offormulations after 5 days at 40° C_(max) (and at time=0). Samples areafter 5 days at 40° C. unless marked as time=0. Formulations from leftto right. FIGS. 2A and 2B show SDS-PAGE analysis of, from left to right,(molecular weight marker) MWM, 3, 4, 5, 8, 9, 10, 11, 13, 14, 15, 16,17, 19, reference. FIG. 2A was performed with a reducing agent and FIG.2B was performed without a reducing agent. FIG. 2C shows SDS-PAGEanalysis of, from left to right, 1 t0, 3 t0, 5 t0, 1, 2, 3, reference,MWM, 1 t0, 3 t0, 5 t0, 1, 2, 3, reference. For FIG. 2C, lanes to theleft of MWM were performed with a reducing agent and lanes to the rightof MWM were performed without a reducing agent. Reference=Pilot Lot,Sublot #2 (−20° C.). MWM (top to bottom): 188k, 98k, 62k, 49k, 38k, 28k,17k, 14k, 6k, 3k. Black arrows point to higher molecular weight species,green arrow points to new band in formulation 19, and red arrow pointsto what may be reduced material in formulations 5 and 15.

After 3 days at room temperature in the light, higher molecular weightspecies formation was evident (FIG. 3 ). Samples are from after 3 daysat room temperature in the light unless marked as F/T. FIGS. 2A and 2Bshow SDS-PAGE analysis of, from left to right, (molecular weight marker)MWM, reference, 3, 4, 5, 8, 9, 10, 11, 13, 14, 15, 16, 17, 19. FIG. 3Awas performed with a reducing agent and FIG. 3B was performed without areducing agent. FIG. 3C shows SDS-PAGE analysis of, from left to right,3 F/T, 13 F/T, 14 F/T, 1, 2, 3, reference, MWM, 3 F/T, 13 F/T, 14 F/T,1, 2, 3, reference. For FIG. 3C, lanes to the left of MWM were performedwith a reducing agent and lanes to the right of MWM were performedwithout a reducing agent. Reference=Compound 16, Pilot Lot, Sublot #2(−20° C.). MWM (top to bottom): 188k, 98k, 62k, 49k, 38k, 28k, 17k, 14k,6k, 3k. Black arrows point to HMWS.

The effect of various excipients on Compound 16 stability was testedwith Compound 16 in a base buffer. Formulations were stored inmicrocentrifuge tubes with an air atmosphere and assayed after 2 daysand 5 days at 40° C. in the dark, after 3 days at room temperature (rt)in the dark or in ambient light, and after 3× freeze/thaw between −20°C. and room temperature in the dark.

Example 3 Evaluation of Chlorotoxin Conjugate Stability in Various VialTypes and Atmospheres

This example describes the stability of Compound 16 in different vialtypes and in different atmospheric (headspace) environments.

The stability of KNTi-0303 (the active pharmaceutical ingredient ofBLZ-100) was tested in amber glass, clear glass, CZ, and Type 1+glassvials with air or N₂ headspace. The purity of samples was assessed byRP-HPLC, as shown in Table 31. All samples degraded significantly uponincubation at 40° C. In some cases, the purity values after 8 d at 40°C. were similar or even higher than the values after 5 d at 40° C. Boththe 5 d and 8 d samples were reanalyzed together the following week, andthe purity values as well as chromatogram shapes were comparable to theoriginal analyses. The 2 d, 5 d, and 8 d vials were all in the 40° C.incubator on the same days, with the vials being removed after the setnumber of days, placed at 5° C. in the dark, and then analyzed within 1d.

TABLE 31 % purity by RP-HPLC of Compound 16 in stored in different vialtypes. % purity RP-HPLC Configuration 2 d 5 d 8 d 1 d 3 d 1 d 3 d 1 d rt3 d rt 3X # Vial Atm t0′ 40 C. 40 C. 40 C. t0 rt lt rt lt rt dk rt dkshake shake F/T 1 Amber Air 98.7 92.2 72.6 72.9 98.8 98.2 96.8 98.0 97.6nt nt nt 2 Amber N₂ 98.7 94.2 75.4 80.2 98.8 98.3 98.0 98.8 98.2 98.597.7 98.4 3 Clear Air 98.7 91.1 74.3 70.6 98.8 97.8 95.8 nt nt nt nt nt4 Clear N₂ 98.7 95.5 85.5 84.7 98.8 98.4 97.6 nt nt nt 98.0 98.9 5 CZAir 98.7 93.3 74.3 72.4 98.8 97.6 95.5 nt nt nt nt nt 6 CZ N₂ 98.7 95.983.1 79.6 98.8 98.3 96.7 nt nt nt 97.7 98.9 7 Type 1+ Air 98.7 93.8 76.272.3 98.8 97.6 95.8 nt nt nt nt nt 8 Type 1+ N₂ 98.7 95.5 85.8 80.8 98.898.1 97.5 nt nt nt 98.3 98.8

In all vials stored at 40° C., purity loss by RP-HPLC was significantlyslower when samples were stored under N₂ rather than air. Under N₂,purity loss was significantly more rapid in amber glass vials than inclear glass, Type 1+ glass, or CZ vials. Under air, however, there wasnot a clear difference between vial types, though degradation may havebeen slower in Type 1+ glass or CZ vials.

When exposed to light, purity loss by RP-HPLC in all vials was againsignificantly slower when samples were stored under N₂ rather than air.With light exposure, purity loss was slowed by storage in amber glassvials rather than the other vial types. Under N₂, purity loss after 3 din the light (8 h of light exposure per day) was 0.8% in amber glassvials and 1.2% in clear glass vials. Under air, purity loss after 3 d inthe light was 2.0% in amber glass vials and 3.0% in clear glass vials.Thus, the improved purity by storage in amber vials after 3 d in thelight is 0.4% under N₂ and 1.0% in air compared to clear glass vials.Clear glass vials, Type 1+ vials, and CZ vials performed similarly withlight exposure except that CZ vials under N₂ were less protective thanother vials under N₂, likely due to the increased gas permeability of CZvials.

Comparing data from samples at rt in the light versus in the dark showsthat the formulation in amber vials undergoes very little degradationdue to light exposure over 3 d. After 3 d, the additional purity lossfrom light exposure was 0.2% under N₂ and 0.8% in air.

By RP-HPLC, Compound 16 did not appear very sensitive to shaking in theformulations and configurations tested. Compared to rt storage in thedark without shaking, there was an additional purity loss of 0.5% due toshaking for 3 d in amber vials under N₂. There was no clear differencebetween vial types upon shaking under N2, but the stability may beslightly higher in clear glass or Type 1+ glass vials Shaking providesagitation stress but also provides additional convection that couldincrease exposure to oxygen in the headspace or surface leaching.

There was no apparent degradation after 3× F/T in any vial type under N₂as assessed by RP-HPLC. While the value appears slightly lower for theamber vials, the 1^(st) RP-HPLC injection yielded a value of 98.0% butthe 2^(nd) injection yielded a value of 98.8%. The 2^(nd) injection wascomparable to the other vial types; it is possible the 1^(st) injectionwas artificially low due to effect of the prior injected sample.

Example 4 Development of Candidate Formulations for LyophilizedConjugates

This example shows various lyophilization conditions for Compound 16.

Methods: Various strategies for lyophilization of Compound 16 wereattempted for different formulations of Compound 16. For freezingtechniques, 1.2 mL of a formulation was added to 3 mL glass vial andstoppered with single vent fluorotec coated stopper. For fast freezing,vials were placed on a shelf after equilibration of the shelf at −40° C.For slow freezing, the temperature was controlled to reduce from 4° C.to −40° C. at a rate of 1° C./min. The peptide-dye conjugateconcentrations used were from 1, 3, 4, 6, and 10 mg/mL.

Lyophilized Compound 16 was reconstituted by addition of 1.2 mL of waterand followed with gentle mixing for <1 min. The reconstituted Compound16 was a clear, emerald green solution with no visible particles and nopellet observed after centrifugation (15,000×g, 5 min). The pH rangedfrom 6.8-6.95 and the moisture content was <1% as determined by KFtitration.

Prior to lyophilization, the formulation volumes were each 1.2 mL andlyophilized formulations were reconstituted with 1.1 mL of waterfollowed by gentle mixing for <1 min. No visible aggregates wereobserved during or after reconstitution.

Reconstituted samples were analyzed for oxidation/purity by RP-HPLC,mass determined by RP-HPLC and/or OD786, aggregation measured by pelletformation after centrifugation, MFI and/or SDS-PAGE, chargeheterogeneity determined by cIEF, secondary structure indicated by FTIR,pH measurements as described herein (the pH ranged from 6.8-7.0) and theKF titration determined (residual moisture was <0.5%).

Results: Summary data for various lyophilized Compound 16 formulationsare provided in Tables 32-33. As determined by RP-HPLC, lyophilizationwith fast and slow freezing produced no substantial differences in totalpeak area of chromatograms from formulations with 8% Mannitol and 2%trehalose (FIG. 4 ). The FTIR spectra of reconstituted samples werecomparable to FTIR spectra of samples that had not been lyophilized(Table 33). Secondary structures indicated by FTIR spectra did notdiffer between formulations that underwent fast versus slow freezing(FIG. 5 ). No difference in particle size was detected by SDS-PAGE ofvarious lyophilized formulations (FIG. 6 ). There also was nosignificant difference in charge heterogeniety between samples(isoelectric point, pI), as determined by cIEF.

TABLE 32 Conservative Lyophilization Cycle Freeze Primary/SecondaryDrying Final Step 1 1 2 3 4 5 6 7 1 Shelf Temp −40 −40 −30 −35 −20 −10 020 4 (° C.) Ramp Rate — 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1.0 (° C./min) Time(min) 540 270 400 920 600 60 60 60 HOLD Vacuum — 100 100 100 100 100 100100 100 (mT)

TABLE 33 Properties of Formulations. Pellet OD cIEF RP-HPLC API ConcCake Intr- Obs SDS 786 Area Purity # (mg/mL) Formulation Appearance pHFTIR FL (Y/N) Page (%) (%) (%) 1 3 No Lyo N/A 6.96 NC NC N NC 98.1 95.098.9 2 3 8% Mannitol Intact 6.75 NC NC N NC 112.8 95.3 98.2 2% Trehalose3 3 8% Mannitol Intact 6.69 NC NC N NC 118.5 95.6 98.4 2% Trehalose 10mM Met 4 3 8% Mannitol Intact 6.93 NC NC N NC 113.0 93.8 98.4 2%Trehalose 20 mM Met 5 3 8% Mannitol Intact 6.97 NC NC N NC 116.0 92.896.8 2% Trehalose 0.05% PS20 6 3 8% Mannitol Intact 6.91 NC NC N NC115.1 94.6 96.8 2% Trehalose 0.05% PS80 7 3 8% Mannitol Intact 6.84 NCNC N NC 126.4 93.7 97.2 2% Trehalose 10 mM Met, 0.05% PS20 8 3 10%Mannitol Intact 6.77 NC NC N NC 119.4 91.8 97.1 10 mM Met, 0.05% PS20 93 5% Mannitol Intact 6.78 NC NC N NC 106.7 91.3 97.2 10 mM Met, 0.05%PS20 10 3 4% Mannitol Intact 6.90 NC NC N NC 114.4 90.0 97.3 2%Trehalose 10 mM Met, 0.05% PS20 11 3 10% Mannitol Intact 7.08 NC NC N NC80.6 95.5 98.5 12 1 5% Mannitol Intact 7.03 NC NC Y NC 99.0 88.9 92.10.05% PS20 1 mg/mL 13 4 5% Mannitol Intact 6.96 NC NC N NC 83.0 91.497.1 0.05% PS20 4 mg/mL 14 7 5% Mannitol Intact 6.94 NC NC N NC 87.091.4 97.8 0.05% PS20 7 mg/mL 15 10 5% Mannitol Intact 6.73 NC NC N NC84.0 90.4 98.1 0.05% PS20 10 mg/mL 16 6 5% Mannitol N/A 6.91 NC NC N NC92.0 92.4 98.9 No Lyo 17 6 5% Mannitol N/A 7.08 NC NC N NC 156.0 92.298.9 0.05% PS20 No Lyo 18 6 5% Mannitol N/A 7.15 NC NC N NC 104.0 92.298.9 10% mM Met No Lyo 19 6 5% Mannitol N/A 7.12 NC NC N NC 100.0 92.498.9 10 mM Met 0.05% PS20, No Lyo 20 6 5% Mannitol Intact 7.08 NC NC NNC 113.5 90.5 98.9 21 6 5% Mannitol Intact 7.06 NC NC N NC 135.0 89.998.9 0.05% PS20 22 6 5% Mannitol Intact 7.13 NC NC N NC 97.0 90.2 98.810 mM Met 23 6 5% Mannitol Intact 7.04 NC NC N NC 97.5 87.5 98.9 10 mMMet 0.05% PS20 24 3 7% Mannitol Intact 6.81 ND ND N ND 82.0 ND ND 3%Trehalose 25 3 9% Mannitol Intact 6.93 ND ND N ND 81.0 ND ND 1%Trehalose 26 3 10% Trehalose Phase ND ND ND ND ND ND ND ND separation 273 10% Sucrose Collapse ND ND ND ND ND ND ND ND and Phase Separation 28 310% Trehalose Collapse ND ND ND ND ND ND ND ND 1% Dextran T40 and PhaseSeparation 29 3 8% Trehalose Collapse ND ND ND ND ND ND ND ND 1% Glycineand Phase Separation 30 3 10% Trehalose, Aggregation ND ND ND ND ND NDND ND 1% HSA upon HSA addition 31 3 2% Mannitol Phase ND ND ND ND ND NDND ND 8% Trehalose Separation Intr-FL = Intrinsic fluorescence; N/A =Not applicable; ND = Not Done; NC = No change from control.

Example 5 Preparation and Use of Compound for Targeting of Glioma TumorTissues

This example shows the performance of ICG in targeting tumor tissuecompared to normal tissue, often as the signal/noise ratio and thebiodistribution of ICG conjugates 24 hours after injection into asubject.

N-terminal PEGylation of chlorotoxin (CTX) and Compound 76 prior toconjugation with ICG minimizes product heterogeneity resulting frompotential ICG conjugation to the N-terminus in addition to the K27 site(the peptide sequence of Compound 76 having a sequence ofH-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH).PEGylation often improves solubility in aqueous solutions and the signalto noise ratio during imaging of cancerous tissue.

Three derivatives of Compound 76 were prepared, i.e., PEGaldehydederivatives of 2kD, 5kD, and 40kD. These were obtained by PEGylatingCompound 76 using 2kD, 5kD and 40kD PEGaldehyde derivatives,respectively. 5kD-PEGylated modified chlorotoxin (Compound 76-5kD) wasobtained by PEGylating Compound 76 using a 5kD polydisperse PEGaldehydederivative.

Mice bearing U87 flank xenografts were injected with 2 nmol of eachchlorotoxin based conjugate through the tail vein (IV). Twenty fourhours after injection, the mice were euthanized and the tumor and legmuscle were resected or the brain, heart, liver, kidney, spleen, andblood were collected. Tumor and leg muscle resected tissue was imagedwith the IVIS Spectrum equipped with a 745 nm excitation filter and an820 nm emission filter. All tissue was frozen in OCT and stored.

Tissues were sliced using a cryostat into 12 μm thick sections andscanned using the Li-Cor Odyssey set to 21 μm resolution, high quality,intensity 7.0, channel 800 nm (excitation=785 nm). The images werequantified using the Li-Cor Odyssey software using regions of interest(ROI's) of identical size and expressed in units of Integrated Intensity(counts per mm²). Tissue was submitted to the FHCRC ExperimentalHistopathology core for H&E staining.

Free dye experiments were performed using Cardiogreen (ICG) (Sigma,product # I 2633). A 200 μM solution was prepared in H₂O and filtersterilized. A 40 μM dilution was prepared in 1× PBS. The ICG free dyeremained in solution after this procedure and was used within 1 h afterreconstitution.

Results. The Odyssey analysis showed signal/noise for each conjugate asfollows: Compound 76-2kD, 6.6+/−3.7 (n=8); Compound 76-5kD, 8.8+/−1.6(n=7); Compound 76-40kD, 10.3 +/−4.4 (n=8). Representative fluorescentimages from each of the conjugates are shown (FIG. 8 ). The capsulesurrounding the tumor tissue has a high level of signal compared to thetumor cells (FIG. 9 ).

Compound 76-2kD, Compound 76-5kD, Compound 76-40kD targeted the tumortissue. The free dye is not detected in the tumor 24 hours afterinjection. PEGylated chlorotoxin conjugated to the fluorophore ICG bindsto tumor tissue and is detected 24 h after injection. Tumor binding isspecific to the properties of PEGylated chlorotoxin as the free dye wasnot detectable in tumors after 24 h.

Biodistribution patterns 24 h after injection show that Compound 76-5kDdistributes mainly to the liver and kidney with a low signal detected inthe spleen and heart. Little or no signal was detected in normal brain(FIG. 10 ). Additionally, significant signal was not detected in thegreat vessels of the heart, which has been observed with the IR800 andCy5.5 conjugates in past experiments (FIG. 11 ).

ICG free dye is not detectable in liver, kidney, spleen, or brain. Freedye is detectable in the fecal matter indicating that it is cleared fromthe blood by the liver in into the bile. The biodistribution patterns ofthe conjugates therefore more closely resembles that which is seen forother CTX conjugates while the free dye biodistribution is similar towhat is reported for non-conjugated ICG.

Example 6 Administration of Other Chlorotoxin Conjugate Compounds andTargeting of Glioma Tumor Tissues

This example shows the use of Compounds 1-720 in targeting tumor tissuecompared to normal tissue, as the signal/noise ratio and thebiodistribution of chlorotoxin conjugate compounds 1 to 720 at 24 hoursafter injection into a subject.

Materials and methods are used as described in Example 5 but withCompounds 1-720.

Results. Compounds 1-720 preferentially target and label tumor tissueand capsules surrounding tumors. Biodistribution patterns 24 h afterinjection show that Compounds 1-720 distribute mainly to the liver andkidney with a low signal detected in the spleen and heart. Little or nosignal is detected in normal brain or in great vessels of the heart.

Example 7 Dose Intervals and Imaging Intervals Using Compound 16 forDetection of Glioma Tumors in Models

This example shows the optimal imaging time and dose of Compound 16 inmice and further shows that Compound 16 targets U87 human glioma cellsimplanted into the brains of mice. Compound 16 signal in tumor comparedto normal brain (signal-to-noise ratio, SNR) was compared to the SNRcalculated in subcutaneous U87 flank xenografts using both whole tissueimaging and sliced tissue analysis.

Materials. Compound 16 in 10 mM Tris and 5% Mannitol at concentrationsof 0.03 mg/ml or 6 μM (0.6 nmole/100 μl); 0.1 mg/ml or 20 μM (2nmole/100 μl); 0.3 mg/ml or 60 μM (6 nmole/100 μl); 0.5 mg/ml or 100 μM(10 nmole/100 μl); and 1 mg/ml or 200 μM (20 nmole/100 μl); 10 nmole/100μl (0.5 mg/ml).

Methods. Nu/Nu female mice bearing U87 (human glioma cell line culturedusing standard culture conditions in DMEM (Invitrogen), 10% Fetal BovineSerum (Qualified #26140, Invitrogen), Pen/Strep (Invitrogen) xenograftsin the flank were injected through the tail vein with 0.6, 2, 6, 10, or20 nmol of Compound 16 in a total volume of 100 μl. Mice were euthanized1, 2, or 3 days after injection. Tumor, muscle, and skin were collectedfor all mice. Brain, heart, liver, and kidney were dissected for asubset of mice. The tissues were imaged using the IVIS Spectrum (PerkinElmer) and quantified using Living Image software (Perkin Elmer). Thetumor tissue was frozen on dry ice in OCT, sliced into 12 μm sections,and scanned on the Odyssey CLx near- infrared imaging system (Li-CorBiosciences) using the 800 nm channel (785 nm excitation). The tissuewas scanned using the “auto” intensity setting and 21 μm resolution.Images were analyzed using the Image Studio software (Li-Cor) bymeasuring the fluorescent signal within a region of interest (ROI) drawnin each tissue image.

Orthotopic xenograft implants were generated using five week old femaleNu/Nu mice (Harlan Laboratories) anesthetized with isoflurane. The scalpwas swabbed with providone-iodine and alcohol. Using a scalpel, anincision was made in the scalp down the midline in the area of thecerebral cortex. A burr hole was drilled through the skull using amicro-drill fitted with a 0.9 mm bit. The burr hole was placed into theright cerebral hemisphere approximately 1 mm lateral (right) of thesagittal suture, 2 mm anterior to the lambdoid suture, and 2 mm deep.Using a p20 pipet and tip, 100,000 U87 cells suspended in serum-freeDMEM (Invitrogen) was injected into the brain. The burr hole was coveredwith a Gelfoam Sponge (Pfizer) fragment and the incision closed withVetbond tissue adhesive (3M). The mouse received bupivacaine at theinjection site for pain relief Mice developed tumors approximately 4weeks after implantation.

Three mice with orthotopic U87 brain tumors and three mice with U87flank tumors received tail vein injections of 100)11 of a 10 nmo¹/₁00 μldose of Compound 16. One day after injection the mice were euthanizedusing CO₂ inhalation. The brains bearing orthotopic xenografts, flanktumors, and normal brain were excised and imaged on the Ivis Spectrum(Perkin Elmer) using the 745 nm excitation and 820 nm emission filters.The whole tissues were then imaged on the Odyssey CLx near- infraredimaging system (Li-Cor Biosciences) using the 800 nm channel (785 nmexcitation). The tumor tissue was frozen on dry ice in Optimal CuttingTissue medium (OCT) (Tissue Tek), sliced into 12 μm sections, placed oncharged slides (Fisherbrand) and scanned on the Odyssey. The tissue wasscanned using the “auto” intensity setting and 21 μm resolution. Imageswere analyzed using the Image Studio software (Li-Cor) by measuring thefluorescent signal within a region of interest (ROI) drawn in eachtissue image. Slides were stained with Hematoxylin and Eosin (H&E) usingstandard histological protocols.

Results. Signal in tumor compared to muscle (SNR) was analyzed 1-3 daysafter Compound 16 injections. In the flank model, SNR at the one daytime point was greatest with a 6 nmol dose while SNR was highest at thethree day time point with a 20 nmol dose (FIG. 12 ). SNR was driven bythe lower signal in normal muscle which was lower in the 6 nmol cohortthan the 20 nmol group one day after injection (FIG. 12 ). The signal inmuscle decreased considerably three days after injection. Signal in thetumor decreased after three days while the ratio of signal in tumor tomuscle improved.

Normal brain, skin, heart, liver, and kidney were evaluated in a subsetof animals. The tissues were excised one or three days after injectionand imaged ex vivo using the IVIS Spectrum. Signal increased withincreased dose for all tissues. Signal was highest in the skin, liver,and kidney.

Signal declined in all tissues by the three day time point (FIG.13A-FIG. 13C). Signal in the kidney declined 5.6 fold in the 20 nmolgroup while signal in the skin declined by 3.7 fold. Whole body liveanimal imaging shows rapid distribution of Compound 16 six hours afterinjection then a decline signal over the next three days (FIG. 14 ).

In order to standardize tissue thickness, the tissue was sliced in 12 μmsections, scanned, and analyzed. SNR in the orthotopic samples was12.7-200 while the SNR for the flank xenograft tissue was 246-344. Thehigher SNR ratios between whole tissue and sliced tissue analysis ismost likely due to the very low levels of signal detected in normalbrain tissue which were often below the level of detection in 12 μmsections.

Signal in tumor tissue compared to normal muscle was dose and timedependent. The optimal dose for a one day imaging time point wasdetermined to be 6 nmol while the optimal dose for a three day timepoint was 20 nmol. Signal in normal tissues distributes rapidly afterinjection with the highest accumulation of Compound 16 in the kidney,liver, and skin one day after injection. Compound 16 cleared out ofnormal tissues with residual amounts remaining in the kidney, liver, andskin after three days.

Optimal imaging time after Compound 16 injection was assessed for dosesbetween 0.6- and 20 nmol. Mice with U87 flank xenografts were imaged 1,2 or 3 days after injection. For one day imaging, signal in tumorcompared to muscle (signal to noise ratio, SNR) was greatest at 6 nmoldoses. For three day imaging, SNR was greatest at 20 nmol doses.

Tumor targeting and imaging efficacy of Compound 16 was assessed in theU87 orthotopic brain xenograft mouse model of glioma. Mice with U87human glioma cells implanted in either the brain or the flank wereinjected with 10 nmole of Compound 16. Brain and tumor tissue wasexcised and imaged 1 day after injection. Signal in tumor compared tonormal brain was assessed on both whole tissue images and frozen tissuesliced in 12 μm sections. Signal in tumor compared to normal brain (SNR)was 11.6-60 for the orthotopic xenograft samples and 131-138 for theflank xenograft samples (FIG. 13A-FIG. 13C). Because most of the tumortissue adhered to the skull, orthotopic sample #1 was not included inthe quantitative analysis. Residual tumor cells were detected in thebrain (FIG. 13A-FIG. 13C).

Compound 16 was detected in tumors from two out of three U87 orthotopicbrain tumors using the IVIS Spectrum (FIG. 15 ). The brain tumor fromorthotopic #1 did not have a signal on the Ivis Spectrum. The absence ofsignal was due to the tumor tissue adhering to the underside of theskull which was subsequently pulled out of the brain during necropsy.Compound 16 signal was detected in the skull during whole tissue imagingon the Odyssey (FIG. 15 ).

Example 8 Dose Intervals and Imaging Intervals Using Other ChlorotoxinConjugate Compounds for Detection of Glioma Tumors in Models

This example evaluates the optimal imaging time and dose of Compounds1-720 in mice and further shows that Compounds 1-720 target U87 humanglioma cells implanted into the brains of mice. Compounds 1-720 signalin tumor compared to normal brain (SNR) is compared to the SNRcalculated in subcutaneous U87 flank xenografts using both whole tissueimaging and sliced tissue analysis.

Materials and methods are as described in Example 7.

Results. Signal in tumor compared to muscle (SNR) is analyzed 1-3 daysafter Compounds 1-720 injections. SNR at the one day time point isgreatest with a 6 nmol dose while SNR is highest at the three day timepoint with a 20 nmol dose. SNR is driven by the lower signal in normalmuscle which is lower in the 6 nmol cohort than the 20 nmol group oneday after injection. The signal in muscle decreased considerably threedays after injection. Signal in the tumor decreases after three dayswhile the ratio of signal in tumor to muscle improves.

Normal brain, skin, heart, liver, and kidney are evaluated. Signalincreases with increased dose for all tissues. Signal is highest in theskin, liver, and kidney. Histopathology analysis of mice, rats, andnon-human primates treated with doses as high as 100× standard imagingdose of Compounds 1-720 finds no test article related toxicity.

Signal declines in all tissues by the three day time point. Whole bodylive animal imaging shows rapid distribution of Compounds 1-720 sixhours after injection then a decline signal over the next three days.

Signal in tumor tissue compared to normal muscle was dose and timedependent. The optimal dose for a one day imaging time point wasdetermined to be 6 nmol while the optimal dose for a three day timepoint was 20 nmol. Signal in normal tissues distributes rapidly afterinjection with the highest accumulation of Compounds 1-720 in thekidney, liver, and skin one day after injection. Compounds 1-720 clearout of normal tissues with residual amounts remaining in the kidney,liver, and skin after three days.

Optimal imaging time after injection of Compounds 1-720 is assessed fordoses between 0.6- and 20 nmol. Mice with U87 flank xenografts areimaged 1, 2 or 3 days after injection. For one day imaging, signal intumor compared to muscle (signal to noise ratio, SNR) is greatest at alower dose than the optimal dose for high SNR when imaging after threedays.

Tumor targeting and imaging efficacy of Compounds 1-720 is assessed inthe U87 orthotopic brain xenograft mouse model of glioma. Mice with U87human glioma cells implanted in either the brain or the flank areinjected with 10 nmole of Compounds 1-720. Brain and tumor tissue isexcised and imaged 1 day after injection. Signal in tumor compared tonormal brain is assessed on both whole tissue images and frozen tissuesliced in 12 μm sections. Signal in orthotropic brain tumors is higherthan normal brain tissue using whole tissue imaging and sliced tissueanalysis. Signal in flank tumors is higher than normal brain tissueusing whole tissue imaging and sliced tissue analysis.

Example 9 Ex Vivo Image Analysis and Determination of Optimal Dose forImaging in Dogs

This example describes the determination of optimal BLZ-100 imaging dosein dogs with naturally occurring tumors. This analysis was conductedusing tissues from dogs enrolled in the study described in Example 19.The optimal clinical imaging dose is a function of the overallfluorescence intensity of the tumor, which impacts ease of detection,and the ratio of signal in the tumor tissue compared with the normaltissue in which it resides. Comparison of tumor intensity and signal tobackground were performed using ex vivo imaging on gross tumors andsections.

BLZ-100 was given intravenously at fixed doses of 0.1-1.5 mg in a doseescalation scheme, followed by expansion at the apparent optimal dose.To normalize for variation in body size, doses were computed in mg/m²for all dogs. Doses (mg/m²) were 0.25-0.8 (7 dogs), 0.8-1.2 (16) and1.2-1.6 (5).

The Odyssey near-infrared scanner (Li-Cor) is a flat-bed scanner that isoptimized for detection of 800 nm fluorescence. It has 21 micronresolution and gives quantitative data for up to 9 orders of magnitudeof intensity. This instrument was able to measure fluorescence in eventhe very early samples from patients treated with the lowest doses. Datafrom the gross tumor Odyssey scans was used to compare overallfluorescence across all doses administered. The analysis shows that atthe low doses, tumor intensity is correlated with dose level. At dosesup to 0.8 mg/m², signal in gross tumor samples increased as a functionof dose. At doses above 0.8 mg/m², no further gain in fluorescence wasapparent under these conditions. Therefore this is the lower limit ofthe optimal dose range for imaging canine tumors under at least theseconditions.

Tumor type was the most important variable in imaging intensity at dosesgreater than 0.8 mg/m². A total of 21 dogs were treated with doses abovethis threshold. In order to compare gross fluorescence intensity acrosssamples, a region of interest analysis was conducted using the Odysseyscans of the gross tumors. A subset of the soft tissue sarcomas hadhighest overall uptake, followed by the carcinomas (adenocarcinoma andsquamous cell carcinoma). Oral fibrosarcomas had the lowest overalluptake, but it was unclear whether this was due to the histologicsubtype or to anatomic location. There were no associations betweensignal and other study variables, such as breed and body mass. The datashow that soft tissue sarcomas, as a class, have the highestfluorescence, with a median intensity almost threefold that of thecarcinomas, and a maximum more than 7 fold higher.

In dogs treated with 0.8 mg/m² or higher (21 dogs in total), ratios offluorescence in tumor to normal surrounding tissue ranged from <1 (nospecific signal, 3 dogs) to >200, with good differentiation in severaltumor types including meningioma, carcinomas (lung, thyroid, andmammary), and sarcomas. Highest signals and gross tumor to backgroundratios were seen in a subset of soft tissue sarcomas, suggestingpreferential uptake of the conjugate in these tumor types.

The soft-tissue sarcomas showed the most intense fluorescence on grossimaging, so these tumors were selected to perform a histopathologicanalysis of tumor to background ratio for determination of the upperlimit of the optimal dose range. It is presumed that when tumor uptakeis maximal, further dose administration will result in higher backgroundstaining and reduction in tumor to background ratio (TBR). The analysisof soft-tissue sarcomas showed that this was indeed the case.

Four soft tissue sarcoma cases were available for this analysis(Patients 11, 12, 13, and 19). Tissues were sectioned on a cryostat, and30 micron sections were imaged on the Odyssey scanner. These sections orserial sections were stained with H&E and read by an experthistopathologist who was blinded to the fluorescence data. A grid wasoverlaid on the fluorescence image, and total fluorescence in each gridsquare was measured using Image Studio (Li-Cor) software provided withthe Odyssey scanner. Overlay of the fluorescence image with the scoredH&E image enabled calling of tumor vs. non-tumor for each grid square.The average of the fluorescence intensity across all tumor and non-tumorgrid squares in a section was used to compute TBR for each patient. TheTBR declined with increasing dose across these four tumor samples,indicating that higher doses may contribute to increased backgroundstaining and loss of specificity. The time between dose and imaging mayalso influence the TBR.

Dog 13 (TBR 207) had surgery 48 hours after dose administration, anddogs 11 (TBR 0.44), 12 (TBR 16), and 19 (TBR 4) had surgery 24 hoursafter dose administration. Excluding dog 13, comparison of the TBR forthe 24 hour cases shows the same trend, supporting the overallconclusion.

Taken together, these data suggest that the optimal imaging dose fortumor imaging in dogs is in the 0.8-1.2 mg/m² range.

Example 10 Ex Vivo Image Analysis and Determination of Optimal Dose forImaging Using Compound 16

This example describes the determination of optimal chlorotoxinconjugate Compound 16 imaging dose in dogs with naturally occurringtumors.

Chlorotoxin conjugate Compound 16 was given intravenously at fixed dosesof 0.1-1.5 mg in a dose escalation scheme, followed by expansion at theapparent optimal dose. The total fluorescence within each region ofinterest (ROI) was plotted as a function of dose (FIG. 16 ). A subset ofthe soft tissue sarcomas had highest overall uptake, followed by thecarcinomas (adenocarcinoma and squamous cell carcinoma). Oralfibrosarcomas had the lowest overall uptake, but it was unclear whetherthis was due to the histologic subtype or to anatomic location. Highestsignals and gross tumor to background ratios were seen in a subset ofsoft tissue sarcomas (FIG. 17 ), suggesting preferential uptake of theconjugate in these tumor types.

The soft-tissue sarcomas showed the most intense fluorescence on grossimaging, so these tumors were selected to perform a histopathologicanalysis of tumor to background ratio for determination of the upperlimit of the optimal dose range. The average of the fluorescenceintensity across all tumor and non-tumor grid squares in a section wasused to compute TBR for each patient. The results are shown in FIG. 18 .The TBR declined with increasing dose, indicating that higher doses maycontribute to increased background staining and loss of specificity.

Taken together, these data suggest that the optimal imaging dose fortumor imaging in dogs with Compound 16 is in the 0.8-1.1 mg/m² range.

Example 11 Ex vivo Image Analysis and Determination of Optimal Dose forImaging Using Other Chlorotoxin Conjugate Compounds

This example describes the determination of optimal imaging dose ofCompounds 1-720 in dogs with naturally occurring tumors.

Compounds 1-720 are given intravenously at fixed doses, followed byexpansion at the apparent optimal dose.

Materials and methods are as in Example 9, but with Compounds 1-720.

The analysis shows that at the low doses, tumor intensity is correlatedwith dose level. Tumor type is the most important variable in imagingintensity at higher doses. There are no associations between signal andother study variables, such as breed and body mass.

Soft-tissue sarcomas are selected to perform a histopathologic analysisof tumor to background ratio for determination of the upper limit of theoptimal dose range. When tumor uptake is maximal, further doseadministration results in higher background staining and reduction intumor to background ratio (TBR).

TBR declines with increasing dose for tumor samples, indicating thathigher doses may contribute to increased background staining and loss ofspecificity. The time between dose and imaging may also influence theTBR.

Taken together, these data establish an optimal imaging dose for tumorimaging in dogs with Compounds 1-720.

Example 12 Quantitation of Fluorescence by Type of Tumor

This example describes a method for quantitating the fluorescence forvarious tumor types labeled with BLZ-100. Multiple canine tumor typeswere explored in this study in order to determine whether specific tumortypes are more amenable to imaging with a chlorotoxin conjugate thanothers, and to gain broad experience with tumors arising in variousanatomic locations and tissue types.

As an initial study of gross fluorescence intensity, the highest pixelintensity for each gross tumor sample was plotted as described above.All tumors except the meningioma (see below) were available for thisanalysis. Grouping the tumors by anatomic location showed that for mosttumors, overall signal was related more to dose than to anatomiclocation. The exception was for tumors arising in bone; these wereconsistently low at each dose range, suggesting that there may be sometissue-specific influence on BLZ-100 uptake.

Grouping the tumors by tumor type shows that the soft-tissue sarcomas asa group show the highest fluorescence and the most variability in grossimaging. The soft-tissue sarcomas include a wide variety of histologictypes and grades, so the variability seen in this class may be due tovariation in histology and/or grade. They also can have high intratumorvariability (see below), so some of the variation in the Odyssey datacould be due to the sections of tumor that were sampled. Two patientswith fibrosarcoma in the group treated with effective imaging doses hadtumors arising in the jaw. Since tumors arising in the jaw generally hadpoor uptake, it is unclear whether the anatomic site played a role inthe limited uptake and specificity in these tumors.

The intensity values for soft-tissue sarcomas (all subtypes) and thecarcinomas (adenocarcinomas and squamous cell carcinomas) were comparedfor all cases treated with doses at or above 0.8 mg/m². This analysisrevealed a high level of variability among soft-tissue sarcomas, andweaker but more consistent intensities among the carcinomas.

In dogs treated with 0.8 mg/m² or higher (21 dogs in total), ratios offluorescence in tumor to normal surrounding tissue ranged from <1 (nospecific signal) to >200.

Taken together, the gross tissue imaging data suggest that soft-tissuesarcomas are a tumor type with very high potential for the clinicalutility of a chlorotoxin.

Example 13 Quantitation of Fluorescence by Type of Tumor UsingChlorotoxin Conjugate Compound

This example describes a method for quantitating the fluorescence forvarious tumor types labeled with chlorotoxin conjugate Compound 16.Multiple canine tumor types were explored. The intensity values forsoft-tissue sarcomas (all subtypes) and the carcinomas (adenocarcinomasand squamous cell carcinomas) were compared for all cases treated withdoses at or above 0.8 mg/m². This analysis shows the variability amongsoft-tissue sarcomas, and the lower but relatively consistentintensities among the carcinomas (FIG. 19 ).

Ratios of fluorescence in tumor to normal surrounding tissue ranged from<1 (no specific signal) to >200 as shown in Table 34.

TABLE 34 Summary of gross imaging data for dogs treated with 0.8 mg/m²or higher (N = 21). Tumor type Summary of canine tumors Brain tumorsMeningioma (1): TBR 2.5 Head & Neck cancer Oral squamous cell carcinoma(1): TBR 2-3 Lung cancer Adenocarcinoma (1): TBR 3 Breast cancer Mammarycarcinoma (3): TBR 2.5-9 Mammary sarcoma (1): TBR could not becalculated due to lack of adequate normal tissue. Signal in gross tumorwas very high. Skin cancer Cutaneous squamous cell carcinoma (2): TBR2.5-5 Soft-tissue sarcoma Haemangiopericytoma (1): TBR 33-89 vs. skin,73-257 vs. fat Soft-tissue sarcoma, subtype not specified (3): ,TBR 5-17Spindle cell (1): TBR 2 Fibrosarcoma, jaw (2): TBR 0.5-3 (low uptake intumors, and high background in oral mucosa) Hemangiosarcoma, vertebralbody (1): TBR <1 Chondrosarcoma (1): non-specific; patient had radiationtherapy prior to treatment. Tumor was necrotic and nasal mucosa had highbackground. Other Thyroid carcinoma (2): TBR 2-3 Mastocytoma (1): TBR1.5 TBR: tumor to background ratio

Example 14 Quantitation of Fluorescence by Type of Tumor Using OtherChlorotoxin Conjugate Compounds

This example describes a method for quantitating the fluorescence forvarious tumor types labeled with Compounds 1-720. Multiple tumor typesare explored in this study in order to determine whether specific tumortypes are more amenable to imaging with a chlorotoxin conjugate thanothers, and to gain broad experience with tumors arising in variousanatomic locations and tissue types.

Materials and methods used are as in Example 12 but with Compounds1-720. Grouping the tumors by anatomic location shows that for mosttumors, overall signal is related more to dose than to anatomiclocation.

Grouping the tumors by tumor type shows that the soft-tissue sarcomas asa group show the highest fluorescence and the most variability in grossimaging. The soft-tissue sarcomas include a wide variety of histologictypes and grades, so the variability seen in this class may be due tovariation in histology and/or grade. They also can have high intratumorvariability, so some of the variation in the Odyssey data could be dueto the sections of tumor that were sampled.

The intensity values for soft-tissue sarcomas (all subtypes) and thecarcinomas (adenocarcinomas and squamous cell carcinomas) are comparedfor all cases treated with doses at or above 0.8 mg/m². This analysisshows the variability among soft-tissue sarcomas, and the lower butrelatively consistent intensities among the carcinomas.

Taken together, the gross tissue imaging data suggest that soft-tissuesarcomas are a tumor type with very high potential for the clinicalutility of Compounds 1-720.

Example 15 Tolerability of Compound 16 in Normal Mice

This example shows experimental analysis of tolerability of Compound 16in normal CD-1 mice.

Methods. Compound 16 was formulated in 10 mM Histidine, 5% Dextrose atconcentrations of 5, 0.5, 0.05 mg/ml. Chlorotoxin free peptide (KNT-01)was manufactured by Alamone Laboratory and formulated in 10 mMHistidine, 5% Dextrose at a concentration of 1 mM (200 nmole/200 μl).Compound 76 free peptide (KNT-02) was manufactured by American PeptideCompany and formulated in 10 mM Histidine, 5% Dextrose at aconcentration of 1 mM (200 nmole/200 μl) (the peptide component ofCompound 76 having a sequence ofH-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH). Compound 16 free peptide was manufactured by Bachemand formulated in 10 mM Histidine, 5% Dextrose at concentrations of 1mM, 0.1 mM and 0.01 mM (the peptide component of Compound 16 having asequence ofH-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Arg-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Arg-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH).

6-10 week old female CD-1 mice were injected with 2, 20, or 200 nmol ofCompound 16 diluted in 200)11 of 10 mM Histidine, 5% Dextrose in thetail vein. These dose levels are equivalent to 0.01, 0.1 and 1 mg ofconjugate, respectively. Vehicle only was used as the control group.Mice were observed 10min, 1 hour, and 4 hours after injection and thendaily until euthanasia at 3 or 14 days post dose. Mice were scored usinga modified Body Condition Score (BCS) and activity level by visualinspection. Body weight was measured every 3 days. Blood was collectedusing terminal cardiac puncture and placed in serum separating tubes(SST microtainer). General chemistry screens were performed by PhoenixCentral Laboratory. Major organs (brain, heart, kidney, liver, lungs,intestines, skin, and spleen) were dissected, placed in 10% bufferedformalin, paraffin embedded, stained with hematoxylin and eosin, andevaluated by a board certified veterinary pathologist.

6-10 week female CD-1 mice were injected with 0.008, 0.08, or 0.8 mg(molar equivalent of conjugate) of native chlorotoxin (KNT-01), Compound76 (KNT-02), or Compound 16 (KNT-03) free peptide diluted in 200)11 of10 mM Histidine, 5% Dextrose in the tail vein. The mice were observedfor 1 hour post injection for activity level.

Results. Mice were evaluated 10 minutes, 1 hour, and 4 hours afterinjection then once daily until euthanasia 3 or 14 days post treatment.Mice in the vehicle control and the 0.01 mg dose group were normal atall observation time points. Four out of six mice in the 0.1 mg groupand six out of six animals in the 1 mg dose cohort exhibited a decreasein spontaneous motor activity, somnolence, and prostration approximately1-3 minutes after the injection. Ptosis was noted in some mice. Thehypoactive behavior lasted 30-60 minutes and all mice had completelyrecovered by the 4 hour observation time point. The mice were notunconscious or paralyzed. Breathing remained normal. Coloration remainednormal without signs of cyanosis, red eyes, or lacrimation. The decreasein motor activity behavior lasted approximately 30-60 minutes afterinjection.

In order to ascertain if the decrease in motor activity level was due tothe Compound 16 conjugate, additional mice were injected with the KNT-03free peptide, or the related KNT-01 or KNT-02 free peptides. Similar tothe Compound 16 conjugate, all of the mice injected with free peptidesin the high dose (200 nmo1) showed a decrease in activity level,somnolence, and prostration starting 3 minutes post injection. Thisindicated that the hypoactive effect resulted from the peptide backbone.Because the effect was observed in the free peptide with the nativesequence, the transient hypoactivity was not a novel property created bythe mutated Compound 16 peptide and/or conjugation to the dye. Inaddition, this transient behavior was only observed in mice. Rats andnon-human primates injected with similar high doses of Compound 16 didnot exhibit abnormal activity levels.

With the exception of the transient low activity immediately afterinjection all mice were clinically normal by visual inspection for theduration of the study. All mice had BCS3 scores at each healthobservation indicating the mice were healthy. Mice were weighed everythree days until euthanasia. No dose related changes in body weight wereobserved (FIG. 67 ).

Blood was collected from each mouse after euthanasia, at 3 and 14 dayspost injection. The serum was analyzed with a general chemistry screen.No dose related changes were observed. Table 35 shows serum values forkidney and liver function tests. None of the mice had BUN levels greaterthan 40 mg/dl. Compound 16 was detected in the liver and kidney where itwas eliminated in the urine. These tests indicate that Compound 16 didnot damage the kidney and liver, even at high doses.

TABLE 35 Liver and Kidney Serum Chemistry Analysis Animal BUN CreatinineALT AST GGT ID Group (mg/dl) (mg/dl) (U/L) (U/L) (U/L)  3 Days Control19 0.3 27 75 0 Control 22 0.3 23 72 0 Control 22 0.3 25 65 0 0.01 mg 240.2 30 86 0 0.01 mg 28 0.2 26 86 0 0.01 mg 23 0.4 29 77 0 0.1 mg 23 0.426 76 0 0.1 mg 25 0.3 25 83 0 0.1 mg 29 0.4 28 80 0 1 mg 22 0.3 29 76 01 mg 20 0.2 28 124 0 1 mg 31 0.4 35 74 0 14 Days Control 22 0.3 35 83 0Control 31 0.3 30 120 0 Control 19 0.2 30 89 0 0.01 mg 21 0.2 26 80 00.01 mg 17 0.2 52 97 0 0.01 mg 24 0.2 32 109 0 0.1 mg 23 0.2 34 107 00.1 mg 19 0.2 30 117 0 0.1 mg 23 0.3 59 175 0 1 mg 31 0.2 40 81 0 1 mg23 0.1 39 97 0 1 mg 20 0.2 36 206 0 mean 16 0.3 42 84 3 low 9 0 18 45 0high 24 0.4 71 182 19

Major organs were dissected 3 and 14 days after injection. All tissuewas analyzed by a certified veterinary pathologist. There were nohistologic findings attributed to administration of Compound 16 on day 3or on day 14.

Example 16 Tolerability of Other Chlorotoxin Conjugate Compounds inNormal Mice

This example shows experimental analysis of tolerability of Compounds1-720 in normal CD-1 mice.

Materials and methods are as in Example 15, but with Compounds 1-720.

Results. Mice are evaluated 10 minutes, 1 hour, and 4 hours afterinjection then once daily until euthanasia 3 or 14 days post treatment.Some mice exhibit a decrease in spontaneous motor activity, somnolence,and prostration approximately 1-3 minutes after the injection. In orderto ascertain if the decrease in motor activity level is due to Compounds1-720, additional mice are injected with the free peptides. All of themice injected with free peptides show a decrease in activity level,somnolence, and prostration starting 3 minutes post injection. Thisindicates that the hypoactive effect results from the peptide backbone.Because the effect is observed in the free peptide with the nativesequence, the transient hypoactivity is not a novel property created byCompounds 1-720 and/or conjugation to the dye. In addition, thistransient behavior is only observed in mice. Rats and non-human primatesinjected with similar high doses of Compounds 1-720 do not exhibitabnormal activity levels.

With the exception of the transient low activity immediately afterinjection all mice are clinically normal by visual inspection for theduration of the study. All mice have BCS3 scores at each healthobservation indicating the mice are healthy. Mice are weighed everythree days until euthanasia. No dose related changes in body weight areobserved.

Blood is collected from each mouse after euthanasia, at 3 and 14 dayspost injection. The serum is analyzed with a general chemistry screen.No dose related changes are observed. None of the mice have BUN levelsgreater than 40 mg/dl. Compounds 1-720 are detected in the liver andkidney where they are eliminated in the urine. These tests indicate thatCompounds 1-720 do not damage the kidney and liver, even at high doses.

Major organs are dissected 3 and 14 days after injection. All tissue wasanalyzed by a certified veterinary pathologist. There are no histologicfindings attributed to administration of Compounds 1-720 on day 3 or onday 14.

Example 17 Imaging of Compound 16 in ND2:SmoA1 mice with MedulloblastomaTumors

This example shows targeting and illumination of medulloblastoma tumorsand cells in a ND2:SmoA1 mouse model using Compound 16 and further showsclinical and pathological effects of high doses of Compound 16.

Medulloblastoma is the most common malignant solid tumor in children.Current therapy includes maximal safe surgical resection, irradiation,and chemotherapy. Complete surgical resection of the tumor heavilyinfluences the prognosis of patients with medulloblastoma by conferringa 30% survival improvement over patients with residual disease. Patientswith residual disease are considered high-risk for tumor progression andare treated with higher doses of radiation and chemotherapy. Thesepatients have a greater risk of suffering from the deleteriousside-effects of aggressive treatments while facing a lower chance ofsurviving their disease. The goal of near- complete surgical resectionmust be balanced with surgically accurate tumor removal because damaginghealthy brain tissue could severely impair normal neurologicalfunctions. Strategies, such as Compound 16 guided surgery, are needed toimprove complete and accurate tumor resection in patients with braincancer increasing patient survival and reducing morbidity.

The ND2:SmoA1 (abbreviated SmoA1) mouse model of medulloblastoma on aC57BL/6 background was used to evaluate binding of Compound 16 tomedulloblastoma tumor. These mice develop spontaneous medulloblastomatumors in the cerebellum that closely resemble the human disease. Thesegenetically engineered transgenic mice express a constitutively activesmoothened mutant protein (SmoA1) driven by a 1-kb fragment of theneuroD2 promoter. This promoter is activated mainly in the cerebellargranule neuron precursors in the brain. This mouse model mimics thesonic-hedgehog pathway subtype of medulloblastoma. Symptomatic mice thatwere homozygous for the transgene were selected for enrollment in thesestudies. Clinical symptoms of brain cancer were detected using an openfield cage evaluation. Symptoms include head tilt, hunched posture,ataxia, protruding skull, and weight loss.

To evaluate Compound 16 signal in normal brain, Nu/Nu mice received anintravenous injection of 6 nmol of Compound 16. These mice wereadministered 60 μL of 0.5 mg/ml (10 nmo1/100 μI) through the tail vein(n=6). One day after injection, the mice were euthanized using CO₂inhalation. The normal brain was then removed and imaged using the IVISSpectrum (Perkin Elmer) with the 745 nm/820 nm excitation and emissionfilter set. Signal was analyzed using the Living Image software (PerkinElmer) by drawing equal sized “regions of interest” (ROI's) within thebrain tissue. SNR (signal to noise ratio) was calculated using thenon-injected ROI and the average of the normal injected brains.

SmoA1 mice with moderate symptoms of medulloblastoma received anintravenous injection of 10 nmol of Compound 16. Mice were administered100)11 of 0.5 mg/ml (10 nmo1/100 μl) Compound 16 through the tail vein.One day after injection the mice were euthanized using CO₂ inhalation.Ex vivo whole brain imaging was performed with the IVIS Spectrum (PerkinElmer) using the 745 nm/820 nm excitation and emission filter set, andon the Odyssey CLx (Li-Cor Biosciences) near-infrared scanner using the800 nm setting (785 nm excitation laser). The tissue was then frozen inOptimal Cutting Temperature medium (OCT, Tissue Tek) on dry ice or fixedin 10% neutral buffered formalin. The frozen tissue was sliced into 12μm sections. Tissues were either scanned using the Odyssey CLx scanneror stained with hematoxylin and eosin (H&E) according to standardhistological protocols. For brains that were fixed in 10% neutralbuffered formalin, the tissue was processed, embedded, sliced, and H&Estained according to standard protocols at Histology ConsultationServices. Slides were scanned on the Aperio Scanscope AT (LeicaBiosystems) at 20x magnification.Images were analyzed using eitherLiving Image software (Perkin Elmer) or Image Studio software (Li-CorBiosciences) by measuring the fluorescent signal within a region ofinterest (ROI) drawn in each tissue image. Signal in tumor compared tonormal tissue (SNR) was calculated using tumor and cortex measurementsfrom the same brain. Analysis was performed in both the dorsal andventral orientations due to variations in tumor location within thebrain. Signal measurements from whole tissue ROI's were used forstatistical analysis. Significance values were calculated using aone-tailed T-test of unequal variance.

SmoA1 mice with medulloblastoma were injected with Compound 16 andimaged one day later. Compound 16 was higher in tumor tissue compared tonormal brain in all eight of the SmoA1 mice that were enrolled in thestudy. Signal in tumor tissue compared to normal brain was between 5.9and 47. Four out of eight samples had considerably lower signal in thetumor which was near the lower level of detection for the IVIS Spectrumand the Odyssey scanner. Signal in unaffected cortex was 2.7-3.6 foldhigher and signal in normal brains from Nu/Nu mice was the same asnon-injected animals. Small foci of metastatic leptomeningeal spreadwere detected using Compound 16.

Background signal in normal brain due to non-specific binding orincomplete clearance was evaluated in mice that had been injected with 6nmol of Compound 16. The non-injected animal had a background signal of3.17×10⁷ as expressed by radiant efficiency. One day after Compound 16administration, the average radiant efficiency was 3.26 x 10⁷ +/−2.5×10⁶(n=6) which was not appreciably higher than the brain sample that didnot receive an injection with a SNR of 1.03. Compound 16 wassufficiently cleared from normal brain tissue without non-specificbinding one day after injection.

Compound 16 was analyzed in tumor bearing SmoA1 mice (n=8) with a 10nmol dose one day after injection. Using whole brain analysis on theOdyssey, all eight samples had a higher signal in the tumor than thenormal cortex (p=0.03). The fluorescent signal was noticeably lower infour out of eight tumors and near the lower limit of detection on boththe IVIS Spectrum and the Odyssey scanner (FIG. 20 ). Signal in tumorcompared to normal brain (SNR) was between 5.9 and 47.3 (FIG. 20 ).Signal in the cortex of the SmoA1 brain was 2.7-3.6 fold higher than thenon-injected animal whereas the signal in the Nu/Nu normal brain was thesame as the non-injected animal at the same time point (FIG. 20 ). Thehigher signal was possibly caused by the higher dose of Compound 16 thatwas used in SmoA1 mice or abnormalities that affect the entire SmoA1brain. The SmoA1 mice often have mild to severe hydrocephalus whichoften affects clearance of Compound 16 from non-tumor tissue. pLeptomeningeal spread in SmoA1 mice was occasionally observed. Thesemice, like 30-35% of human patients, develop small foci of tumor cellsin the meningeal membrane. Leptomeningeal spread was detected in onemouse using Compound 16. A whole brain fluorescent scan of SmoA1-1 isshown (FIG. 21 ). The scan illuminates small foci of fluorescence thatcorresponds to small clusters of tumor cells highlighted in the H&Estained slide (FIG. 21 ).

All mice with medulloblastoma had a higher signal in the tumor thannormal cortex with SNR's ranging from 5.9-47 after administration ofCompound 16. While the signal in tumor was higher than normal in allmice, four of the samples had noticeably lower signal in the tissue.Compound 16 signal was not detected in mice with normal brain tissue(Nu/Nu mice) while signal in unaffected SmoA1 cortex was 2.7-3.6 foldhigher than the non-injected animals. The residual background signal innon-tumor tissue is possibly caused by hydrocephalus which occurs oftenin these mice. Small foci of metastatic cells were detected usingCompound 16 in one mouse emphasizing Compound 16's capability to detecteven small clusters of cancerous tissue.

Example 18 Imaging of Other Chlorotoxin Conjugate Compounds in ND2:SmoA1mice with Medulloblastoma Tumors

This example shows targeting and illumination of medulloblastoma tumorsand cells in a ND2:SmoA1 mouse model using Compounds 1-720 and furthershows clinical and pathological effects of high doses of Compounds1-720.

The ND2:SmoA1 (abbreviated SmoA1) mouse model of medulloblastoma on aC57BL/6 background is used to evaluate binding of Compounds 1-720 tomedulloblastoma tumor.

Materials and methods are as in Example 17.

SmoA1 mice with medulloblastoma are injected with Compounds 1-720 andimaged one day later. Compounds 1-720 is higher in tumor tissue comparedto normal brain in all eight of the SmoA1 mice that are enrolled in thestudy. Background signal in normal brain due to non-specific binding orincomplete clearance is evaluated in mice that have been injected with 6nmol of Compounds 1-720. Compounds 1-720 is sufficiently cleared fromnormal brain tissue without non-specific binding one day afterinjection.

Compounds 1-720 analyzed in tumor bearing SmoA1 mice has a higher signalin the tumor than the normal cortex.

Leptomeningeal spread in SmoA1 mice is occasionally observed and isdetected using Compounds 1-720.

Mice with medulloblastoma have a higher signal in the tumor than normalcortex. Compounds 1-720 signal is not detected in mice with normal braintissue (Nu/Nu mice) while signal

Small foci of metastatic cells are detected using Compounds 1-720emphasizing the capability of Compounds 1-720 to detect even smallclusters of cancerous tissue.

Example 19 Detection of Naturally Occurring Solid Tumors in Dogs

This example describes methods for detecting naturally occurring solidtumors in dogs using the fluorescently labeled chlorotoxin conjugate,BLZ-100.

Many types of canine tumors resemble human disease, including sarcomas,breast and lung cancers, mucosal squamous cell cancers, and gliomas. Thediversity of these spontaneously occurring tumors in size and type,surrounding tissue, and patient body mass provides a model that issuperior to the mouse in predicting the clinical characteristics of achlorotoxin conjugate, such as BLZ-100, including tumor penetration,background staining, and effective imaging dose.

Methods:

Twenty-eight dogs undergoing planned solid tumor resection were enrolledin the study. All options were discussed and client consent was obtainedunder an approved IACUC protocol. Dogs received standard of careincluding tumor resection with intent to control or cure local disease.Dogs received BLZ-100 intravenously 24-48 hours before surgery, andtissues were imaged ex vivo after surgery. Ex vivo imaging was performedon gross tissue specimens using the IVIS Spectrum (PerkinElmer) and theOdyssey NIR scanner (Li-Cor) to determine overall signal in tumor andgross signal to background. Tissues were embedded in OCT, sectioned on acryostat, and scanned on the Odyssey. Serial sections were stained withH&E, and comparison with the fluorescence scans was used to validate thespecificity of BLZ-100 for tumor tissue. Intraoperative imaging wasconducted in several cases, using a prototype open NIR imaging device.

Dose Preparation:

BLZ-100 was prepared in a formulation buffer (100 mM histidine/5%dextrose, 10 mM Tris/5% dextrose, or 10 mM Tris/5% mannitol). For alldose batches, lyophilized conjugate was suspended in formulation buffer.The material was drawn up into a sterile syringe, and then aliquotedthrough a sterile 0.44 micron filter into pre-capped sterile amber glassvials. The vials were stored at −20° C., and were shipped on dry ice tothe study site.

Patients and Dose Administration:

Twenty-eight dogs with spontaneously-occurring solid tumors wereenrolled in the study. Tumor types included several subtypes of sarcoma;oral and cutaneous squamous cell carcinomas; mast cell tumors;adenocarcinomas including lung, mammary, and thyroid; and a brainmeningioma.

BLZ-100 was given intravenously 24-48 hours prior to surgery. Completeblood count, serum chemistry, and urinalysis data from prior to, and24-48 hours post injection, were evaluated for changes that mightindicate toxicity from the CTX. There were small declines in serum BUN,calcium, and potassium levels and in urine pH that reached statisticalsignificance (two-tailed t-test); however, they remained well within thenormal ranges and were not considered clinically significant. There wereno other significant differences, and no overt safety concerns noted.

Nearly all dogs experienced an immediate pseudoallergy/hypersensitivityreaction within 10 minutes of dosing, characterized by erythema, mild tosevere pruritus, and less commonly swelling of the muzzle and distalextremities. The severity of the reactions was not related to dose levelor rate of administration. The reactions were self-limiting, amelioratedby diphenhydramine, and were completely resolved within 4 hours in allcases. These observations are consistent with a systemic release ofhistamine, which has been reported for a wide variety of drugs. Somedogs (mast cell tumor and CNS tumor patients) were receivingcorticosteroids as part of their standard management, butcorticosteroids were not required for management of reactions in anypatient. No other systemic changes were identified. All dogs toleratedanesthesia and surgery normally. There were no apparent surgicalcomplications associated with BLZ-100.

Patients 13-17 had surgery 2 days after BLZ-100 administration. This wasdone in order to evaluate the impact of time on tumor to backgroundratio. Since no consistent improvement was seen, the remainder of thepatients had surgery 1 day after BLZ-100 administration.

Pharmacokinetics:

Serum samples were collected at time points following doseadministration. A fluorescence assay was used to calculate the serumconcentration of BLZ-100. The data show a rapid distribution intotissues, followed by a slower elimination phase. These data are similarto those obtained from laboratory mice, rats, dogs, and non-humanprimates.

Example 20 Detection of Naturally Occurring Solid Tumors in Dogs UsingChlorotoxin Conjugate Compounds

This example describes methods for detecting naturally occurring solidtumors in dogs using Compound 16.

Materials and methods used were as in Example 19. Patientcharacteristics, tumor type and site, body weight, and dose informationare summarized in Table 36.

TABLE 36 Summary of tumor characteristics, patient data and doseadministered Age Weight Dose Patient Tumor Type Site Breed Sex (yr) (kg)( mg) 1 Soft-tissue Subcutaneous Labrador M 11.7 24 0.375 sarcoma mix 2Adenocarcinoma Lymph node Poodle mix M 6.5 6.7 0.1 3 FibrosarcomaSubcutaneous Rhodesian F 11.7 39.1 0.3 Ridgeback 4 Hemangiosarcoma JawLabrador M 10.7 30.9 0.3 Retriever 5 Mastocytoma Cutaneous Labrador F 1030.5 0.5 Retriever 6 Mastocytoma Cutaneous Pit Bull F 5.3 26 0.5 Terrier7 Adenocarcinoma Lung American F 11 14.3 0.5 Eskimo 8 Squamous cell JawEnglish M 5 25.8 0.5 Springer Spaniel 9 Chondrosarcoma Nasal Labrador F8.4 29.4 1 mix 10 Adenosquamous Mammary Pit Bull F 7 22.1 0.9 carcinomaTerrier mix 11 Soft-tissue Mammary Yorkshire F 7 3.8 0.4 sarcoma Terrier12 Soft-tissue Subcutaneous Border F 3.9 27.9 1 sarcoma Collie mix 13Hemangiopericytoma Subcutaneous Standard F 7 33.7 1 Poodle 14 Squamouscell Cutaneous Pit Bull F 11.8 28 1 carcinoma Terrier 15 Squamous cellJaw Springer M 8.3 23 1 carcinoma Spaniel 16 Fibrosarcoma Jaw Golden F10.7 39.6 1.5 Retriever 17 Fibrosarcoma Jaw Chesapeake M 5 44.4 1.5 BayRetriever 18 Mastocytoma Cutaneous English F 9 29.3 0.8 Bulldog 19Soft-tissue Subcutaneous Labrador F 10.3 25.6 1 sarcoma Retriever 20Follicular Thyroid Golden F 7 37.7 1 carcinoma Retriever 21Adenocarcinoma Thyroid Boxer F 6.3 25.2 1 22 Adenocarcinoma MammaryBrittany F 7 22.2 1 Spaniel 23 Squamous cell Cutaneous Golden F 9.1 34.41 carcinoma Retriever 24 Adenocarcinoma Mammary Labrador F 13.3 21.80.75 mix 25 Soft-tissue Subcutaneous Golden F 13.1 39 1 sarcomaRetriever 26 Soft-tissue Subcutaneous Chow mix F 6.1 33 1 sarcoma 27Meningioma Brain Border F 13.2 17.3 1 Collie 28 HemangiosarcomaVertebral Golden M 10.8 32 body Retriever

Serum samples were collected at time points following doseadministration. A fluorescence assay was used to calculate the serumconcentration of Compound 16. The data show a rapid distribution intotissues, followed by a slower elimination phase (Table 37).

TABLE 37 Serum Compound 16 levels in dogs, measured by standard curveanalysis of fluorescence at time points following dosing. Serum samplesfrom each time point were diluted 1:1 in formulation buffer. A standardcurve of Compound 16 (10 mcg/ml to 4 ng/ml) in 50% serum/50% formulationbuffer was prepared. Fluorescence was measured on the Odyssey scanner(765 nm excitation/800 nm emission). Serum concentrations of the productwere back-calculated using the standard curve. Nominal time point (hr):0.25 1 4 24 48 Dose Calculated serum BLZ-100 Patient (mg/m²)concentration, ng/ml 1 0.44 520.49 31.51 2.21 1.30 2 0.28 91.78 332.4128.63 1.53 3 0.25 1358.79 105.62 61.22 11.87 4 0.30 140.20 48.15 11.422.91 5 0.50 140.70 42.22 14.96 2.31 6 0.56 161.12 42.53 6.93 1.20 7 0.83262.08 56.84 14.04 1.37 9 1.03 2277.50 103.09 84.62 1.14 10 1.12 634.11206.93 12.78 2.92 11 1.62 1685.95 620.00 1.31 12 1.06 8243.41 716.4617.08 4.92 13 0.94 862.19 149.60 14.84 0.69 14 1.06 1561.45 193.46 20.000.53 15 1.21 1668.98 121.85 20.70 0.95 16 1.26 405.91 93.91 20.39 1.1617 1.17 160.69 164.74 2.89 0.71 18 0.82 2084.98 324.36 29.74 0.81 191.13 268.25 121.42 15.08 1.77 20 0.87 1896.58 232.80 195.14 1.31 21 1.14928.75 23.97 3.28 1.02 22 1.24 1069.51 129.34 17.07 0.74 23 0.9319698.61 1921.64 214.52 12.96 24 0.94 18009.88 514.70 173.52 6.44 250.85 666.06 902.32 60.58 8.33 26 0.95 17754.16 1516.42 231.49 8.48 271.47 3008.52 1183.35 107.49 11.18

Example 21 Detection of Naturally Occurring Solid Tumors in Dogs UsingOther Chlorotoxin Conjugate Compounds

This example describes methods for detecting naturally occurring solidtumors in dogs using Compounds 1-720.

Materials and methods are as in Example 19 but with Compounds 1-720.

Serum samples are collected at time points following doseadministration. A fluorescence assay is used to calculate the serumconcentration of Compounds 1-720. The data show a rapid distributioninto tissues, followed by a slower elimination phase. These data aresimilar for laboratory mice, rats, dogs, and non-human primates.

Example 22 Pharmacokinetics and Tolerability of Compound 16

This example demonstrates the pharmacokinetic (PK) profile of Compound16 following a single intravenous (IV) injection in mice, rats, dogs,and monkeys. Pilot studies performed in all four species were used toestimate exposure and inform the study design of definitive GLP studies.PK samples from these studies were analyzed using research-basedmethods. The PK of Compound 16 following IV administration was evaluatedas part of GLP single dose toxicology studies in rats and monkeys. Inthe GLP studies, serum Compound 16 concentrations were determined usingvalidated LC/MS-based methods.

In this example, Compound 16 was intravenously administered as asingle-use intraoperative fluorescent imaging agent to specificallylabel tumor tissue. Compound 16 was sterilely formulated in a liquid at2 mg/mL in Tris/mannitol buffer at neutral pH. The dye was chemicallylinked via a single lysine residue on the CTX peptide. Compound 16contained no novel excipients or linker molecules. A tabular listing ofthe single-dose nonclinical toxicity studies conducted to supportinitiation of first-in-human (FIH) clinical trials of Compound 16 isprovided in Table 38.

TABLE 38 Tabular Listing of Single-Dose Toxicity Studies of Compound 16Compound 16 Study No. Study Title Testing Facility GLP Dose and RouteStatus RPT0031 Pilot Tolerability of Compound 16 Blaze Bioscience No0.01, 0.1, 1 mg Completed and Unconjugated KNT-03 in CD1 Seattle, WA IVMice 10777 Toxicology and Toxicokinetic Xenometrics No 0.03, 0.3, 1.5 mgCompleted Study of Compound 16 in Male Stilwell, KS IV Sprague DawleyRats 10865 Single-dose Intravenous Toxicity Xenometrics Yes 0, 0.07,0.7, 7 Completed and Toxicokinetic Study of Stilwell, KS mg IV Compound16 in Sprague Dawley Rats BRT Evaluation fo the Effect of Burleson No 0,100, 1000, Results 20140605 Compound 16 on Complement Research 10,000ng/mL pending Activiation and Basophill/Mast Cell TechnologiesActivation using Canine In vitro Morrisville, NC Test Systems 10803Pilot Pharmacokinetics Study of Xenometrics No 1 mg Completed Compound16 Following IV Stilwell, KS IV Administration to Male Beagle Dogs. Thisstudy includes safety evaluation endpoints. 11265 Mechanistic Study OfXenometrics No 1 mg Results Pseudoallergy Response In Male Stilwell, KSIV available, Beagle Dogs From a Single IV draft report Dose Of Compound16 in preparation 10822 Pilot Pharmacokinetics Study of Xenometrics No0.6 mg IV Completed Compound 16 Following IV Stilwell, KS Administrationto Male Non-human Primates (Cynomolgus). This study includes safetyevaluation endpoints. 814.02 A Single Dose 14-Day Intravenous SNBL Yes0, 0.6, 6, 60 mg Draft Toxicity Study of Compound 16 in Seattle, WA IVReport Cynomolgus Monkeys

All species used for nonclinical safety evaluations werepharmacologically relevant for Compound 16 based on a high degree ofsequence homology to the CTX peptide target. The rat and monkey wereselected as the primary species for GLP toxicology studies based on thesequence homology to the CTX peptide target (97 and 100% respectively),suitability of the species for safety assessment (i.e., preference forrat over mouse), and lack of potential confounding effects(pseudoallergy/hypersensitivity reactions have been observed in the dogbut not in the monkey). Toxicology studies were performed in accordancewith Good Laboratory Practice (GLP) regulations. ApplicableInternational Conference on Harmonization (ICH) and Food and DrugAdministration (FDA) guidance documents were referred to during thedevelopment of the compound, most notably ICH S6(R1), S9 and M3(R2) andthe FDA Guidance for Industry document “Developing Medical Imaging Drugand Biological Products Part 1: Conducting Safety Assessments”.

The selection of dose levels and method of administration were based oninitial mouse pharmacology models in which a dose level of 0.01 mgproduced consistent tumor imaging. Since the preferred method of doseadministration in the clinical trials is on a fixed basis (i.e., notadjusted for body weight or surface area), dose levels in thenonclinical safety studies were converted to fixed dose levels usingestimated body surface area for each species. The dose level for humanswas estimated using imaging data in mice and dogs (Table 39).

TABLE 39 Estimated Imaging Dose Levels Across Species Species (~BodySurface Area (BSA) Dose Dose Dose m²: kg body wt) ( mg) (mg/m²) (mg/kg)Mouse 0.01 1.3 0.5 (0.008 m²: 0.02 kg) Rat 0.07 1.8 0.28 (0.039 m²: 0.25kg) Dog 1 1 0.03 (1 m²: 30 kg) Monkey 0.6 2.4 0.2 (0.25 m²: 3kg) Human 31.9 0.05 (1.6 m²: 60 kg)

In single-dose non-GLP pilot studies, mice displayed transientlydecreased spontaneous motor activity, somnolence and prostrationfollowing intraveneous (IV) administration of 0.1 and 1 mg of Compound16. Transient salivation was the only finding in the pilot study in malerats following IV administration of 0.03, 0.3, or 1.5 mg Compound 16 0IV. In male dogs, notable findings in the pilot study includedpseudoallergy/hypersensitivity reactions (i.e., itching/scratching, warmears, etc.) in 2 of 2 treated dogs during or immediately after IVadministration of 1 mg Compound 16. The mechanism of the pseudoallergyhas been further explored in 3 male beagle dogs following IVadministration of 1 mg of Compound 16. A rapid rise in plasma histaminelevels was noted in all dogs, coincident with the appearance of clinicalsigns. No changes in complement level were seen, suggesting Compound 16acted directly on mast cells/basophils in the dog. In the pilot study inmale Cynomolgus monkeys, there were no abnormal clinical observationsfollowing IV administration of 0.6 mg Compound 16.

In single-dose GLP toxicology studies, Compound 16 was well tolerated inrats and monkeys. There were no Compound 16 -related adverse findings.The no observed adverse effect level (NOAEL) was the high dose of 7 mg,approximately 28 mg/kg, in rats and the high dose of 60 mg,approximately 20 mg/kg, in monkeys.

In safety pharmacology studies, there were minimal effects on hERGcurrent amplitude in human embryonic kidney cells following treatmentwith 0.2, 0.6 or 2.0 μM Compound 16 and no effects on cardiovascularparameters or respiratory rate in conscious cynomolgus monkeys inresponse to administration of 0.6, 6 and 60 mg Compound 16. In a tumorimaging pharmacology study with safety evaluation endpoints, most dogswith spontaneous tumors exhibited pseudoallergy/hypersensitivityreactions after IV administration of 0.1 to 1.5 mg Compound 16.

Single-Dose Toxicity:

Pilot Tolerability of Compound 16 and Unconjugated Compound 16 in CD1Mice. The objective of this example was to assess the tolerability ofCompound 16 in groups of female CD-1. Mice (n=3 mice/group/time point)were given a single IV bolus injection of Compound 16 at fixed doselevels of 0.01, 0.1 or 1 mg which was the equivalent to 1, 10 and 100times the tumor imaging dose in mice. Tolerability was assessed byclinical observations, serum chemistry and histopathology of severalmajor organs. Animals were euthanized on study days 3 and 14.

At the dose of 0.01 mg, mice were clinically normal during allobservation time points. A subset of mice at 0.1 mg and all mice at 1 mgexhibited a decrease in spontaneous motor activity, somnolence, andprostration occuring as rapidly as 1 minute post-dose. The hypoactivitylasted approximately 30 to 60 minutes. All mice returned to normal bythe 4 hour observation time point and remained normal for the durationof the study. Similar clinical signs were seen when mice were injectedwith equimolar amounts of the peptide backbone of Compound 16.

Toxicology and Toxicokinetic Study of Compound 16 in Male Sprague DawleyRats. Male rats (n=3/group) were intraveneously administered a singlebolus injection of Compound 16 at fixed dose levels of either 0.03, 0.3,or 1.5 mg. Rats were observed up to 48 hours post-dose and wereeuthanized on day 3 of the study. Tolerability was assessed by clinicalobservations, hematology, serum chemistry and gross pathology.

Transient treatment-related clinical signs were limited to salivation,which was observed at the 0.03 mg dose in 2 rats at 20 minutes and 1hour post-dose, at the 0.3 mg dose in 2 rats at 20 minutes post-dose,and at the 1.5 mg dose in 1 rat at 20 minutes, 1 hour, and 2 hourspost-dose. At the 0.03 mg dose (n=3 rats) and the 0.3 mg dose (n=2rats), clear oral stain was observed 1 or 2 hours post-dose. No cleardose-response relationship was evident and clinical signs were resolvedat 2 hours and before 4 hours post-dose. There were no apparent testarticle-related hematology, clinical chemistry, or gross pathologyfindings at any dose level.

Single Dose Intravenous Toxicity and Toxicokinetic Study of Compound 16in Sprague Dawley Rats. Rats (n=10/sex/group) were intravenouslyadministered a single bolus injection of Compound 16 at fixed doselevels of 0, 0.07, 0.7, or 7 mg. Half of the animals were observed up today 3 of the study and were then euthanized. The remaining animals wereobserved up to day 15 of the study and were then euthanized (Table 40).

TABLE 40 Dose Groups for Single-Dose Toxicology Study in Rats Fixed DoseDose Group No. Animals Test Dose Volume Conc. Sacrifice No. (M/F)Article (mg) (mL/rat) (mg/mL) Day 3 Day 15 A 10/10 Vehicle 0 1.4 05/sex/group 5/sex/group B 10/10 Cmpd 16 0.07 0.35 0.2 5/sex/group5/sex/group C 10/10 Cmpd 16 0.7 0.35 2 5/sex/group 5/sex/group D 10/10Cmpd 16 7 1.4 5 5/sex/group 5/sex/group

Single Dose Intravenous Toxicity and Toxicokinetic Study of Compound 16in Sprague Dawley Rats. There were no Compound 16-related adversefindings at any dose level based on clinical observations (e.g.,including detailed open field assessments), mortality, body weights,food consumption, ophthalmology, clinical pathology (e.g., includinghematology, coagulation, clinical chemistry and urinalysis), organweights and histopathology (e.g., including the injection site). At 0.07mg dose, a slight, transient salivation was seen in 1 male rat duringopen field assessment approximately 15 minutes post-dose. Thisobservation was most likely related to Compound 16; however, no cleardose-response relationship was evident.

At the 7 mg dose, the gross pathology findings were limited to greendiscoloration of the kidneys on days 3 and 15 of the study. As notedabove, there were no Compound 16-related findings on organ weights ormicroscopic findings at any dose level. The findings of greendiscoloration in kidneys are not considered adverse. As such, the NOAELwas considered the high dose of 7 mg, or approximately 28 mg/kg.

Evaluation of the Effect of Compound 16 on Complement Activation andBasophill/Mast Cell Activation using Canine In vitro Test Systems. Bloodfrom beagle dogs (n=5) was used to prepare high complement containerserum. Compound 16, at concentrations of 0 (negative control) 100, 1000,and 10000 ng/mL, was incubated in the dog serum for 30, 60 and 120minutes at 37° C. in 96 well plate format. Cobra venom factor, whichcontains native CTX, was used as a positive control. Complementactivation was measured by converting intact dog C3 to the stable endproduct, human sC5b-9, which was quantified by ELISA. Activation ofcomplement in dog serum by Compound 16was assessed by subtracting thetotal amount of complement at baseline, prior to addition of Compound 16from residual C3 at the various timepoints.

Basophil activation was measured in whole blood samples (n=3) isolatedfrom dogs. Compound 16 at concentrations of 100, 1000, 10000 ng/mL wasincubated with the whole blood samples for 10 or 60 minutes. Anti-canineIgE and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were used aspositive controls. The presence of histamine, as measured by ELISA, inthe blood samples was used to assess basophil activation.

Pilot Pharmacokinetics Study of Compound 16 Following IV Administrationto Male Non-Human Primates (Cynomolgus). Cynomolgus monkeys (n=2males/group) were intravenously administered a single bolus injection ofCompound 16 at a fixed dose level of 0.6 mg. Blood samples werecollected for pharmacokinetics at pre-administration, 0.083, 0.25, 0.5,1, 2, 4, 6, 24, 48, 72 and 96 hours post-dose. Clinical observationswere made at each time point. There were no abnormal clinicalobservations up to 96 hours post-dose

A Single Dose 14-Day Intravenous Toxicity Study of Compound 16 inCynomolgus Monkeys. Cynomolgus monkeys (n=3/sex/group) wereintravenously administered a single bolus of Compound 16 at fixed doselevels of 0, 0.6, 6, or 60 mg and were observed for 14 days (Table 41).Ophthalmic examinations were performed prior to dosing at study day -7and on study day 11. The animals were euthanized on study day 15 andgross post-mortem examinations were performed.

There were no Compound 16-related adverse findings at any dose levelbased on mortality, clinical observations, body weights, ophthalmology,clinical pathology (e.g, including hematology, coagulation, serumchemistry and urinalysis), organ weights and histopathology (e.g.,including the injection site). Neurological and musculoskeletalassessments were additional parameters added to the study and notest-article related changes were noted after once weekly assessments.

TABLE 41 Dose Groups for Single-Dose Toxicology Study in Monkeys GroupNo. Animals| Test Fixed Dose Dose Volume Dose Conc. No. (M/F) Article(mg) (mL) (mg/mL) 1 3/3 Vehicle 0 12 0 2 3/3 Cmpd 16 0.6 3 0.2 3 3/3Cmpd 16 6 3 2 4 3/3 Cmpd 16 60 12 5

At 60 mg, green-colored urine was noted by gross examination in one maleand three females at day 3 of the study day with no corresponding impacton urinary parameters or renal pathology. Urine from all animals wassubjected to an exploratory fluorescence assay, which revealed a dosedependent increase in fluorescent signal intensity on day 3 of the studyin all groups treated with Compound 16 compared to the control groupwhich was not treated with Compound 16. On day 15 of the study, thefluorescent signal was lower compared to day 3, but still detected inmost mid- and high-dose animals, but not the low-dose animals whencompared to the control group. These results suggest drug was present inthe urine, which possibly accounted for the green appearance of urine inthe high dose animals. Therefore the NOAEL was considered to be the highdose of 60 mg or approximately 20 mg/kg.

Single bolus doses of Compound 16 administered intravenously werewell-tolerated in mice, rats and monkeys. Treatment-related changesobserved in mice included decreased spontaneous motor activity,somnolence and prostration at doses of 0.1 and 1 mg Compound 16. Thehypoactivity was transient and occurred from 30 to 60 minutes post-dose.All mice were normal by 4 hours. These changes were not observed to-datein the clinical trials or in species other than mice. Clinical signs oftransient salivation were observed in a pilot study in male rats atdoses of 0.03, 0.3, or 1.5 mg Compound 16 following intravenousadministration. However, salivation was not observed in the subsequentsingle-dose study performed in male and female rats at doses up to 7 mgof Compound 16 by intravenous administration, nor was salivationobserved in any other species. In addition to the toxicity evaluations,no effects on heart rate, blood pressure, respiration rate or ECGtracings were observed in a safety pharmacology study in consciousmonkeys.

In vitro hemolysis and local tolerance studies have not been conducted.Compound 16 is a biotechnology-derived pharmaceutical candidate that isformulated with commonly-used excipients, such as Tris buffer andD-mannitol. Importantly, (1) no treatment-related hematological findingswere identified in the completed nonclinical safety studies or in thepreliminary data from the on-going phase 1 clinical safety study insubjects with skin cancer and (2) no irritation or lesions at theinjection site were identified by macroscopic examination or byhistopathology in the single-dose, intravenous administration, ortoxicology studies in rats and monkeys.

Genetic toxicology studies were not conducted with Compound 16 since itis not expected to react with DNA and the formulation does not containany novel excipients. The dye is attached to the peptide via a stable,covalent amide bond. Nothing in the structure of the peptide, dye orattachment suggests the potential of mutagenicity. In addition, typicalclinical use and exposure to Compound 16 will be of short duration,consisting of a single injection per subject, perhaps a single injectionduring a lifetime, with an estimated human plasma terminal half-life ofapproximately 1-2 days, into subjects clinically diagnosed with cancer.Examination of the chromatograms from the LC/MS pharmacokinetic methodshas not revealed the presence of any major metabolites of Compound 16.

The peptide component of Compound 16 is similar to native CTX. Asynthetic version of the CTX peptide (TM-601) has been studied in miceand marmosets and it was well-tolerated. The NOAEL for TM-601 after asingle IV dose in the mouse was 6.4 mg/kg (highest dose tested) and 2.0mg/kg in the marmoset (highest dose tested). Repeated-dosing for 7 weeksin mice at doses of 2 and 5 mg/kg by intravenous administration resultedin clinical signs of transient ptosis and hypoactivity within 1 hourpost-dose. No effects on hematology or tissue pathology were observed.

The typical doses of ICG vary by indication, but generally range from 25to 50 mg. By comparison, Compound 16 contains roughly 0.15 mg of dye permg of drug product. Compound 16 imaging doses are currently estimated torange from 3 to 12 mg, or 0.45 to 1.8 mg equivalents of ICG.

In the pilot studies, Compound 16 PK profiles following IVadministration demonstrated a bi-exponential decline with a rapidinitial phase and a longer terminal phase in all species. In mice andrats, the terminal phase could not be well defined due to lowconcentrations and study design/assay limitations. However, overallsystemic exposure appeared to be well characterized since the majorityof the systemic exposure was accounted for in the first 4 to 8 hoursfollowing IV administration. In the pilot studies of dogs and monkeys,the apparent t_(1/2) was approximately 55 hours in both species.

Following IV administration of Compound 16 in the rat and monkeysingle-dose GLP toxicology studies, exposure based on C_(max) and C₀increased in an approximately dose-proportional manner across the testedranges. AUC_(0-t) values increased in a dose-proportional or higher thandose-proportional manner. These observations suggested that Compound 16clearance is reduced at higher doses.

At the highest dose group of 60 mg Compound 16 in monkeys, the serumconcentration versus time profiles were adequately defined to estimateadditional PK parameters dependent on characterization of the terminalphase. The overall mean t_(1/2), CL, and Vss values were 33.7hr, 50.6mL/hr, and 211 mL, respectively.

Methods of Analysis:

Analytical Method for Quantitation of Compound 16 in PreclinicalSpecies. Two main approaches have been used to quantify Compound 16 inserum. A fluorescence-based method was used in support of non-GLPstudies in the mouse, dog, and monkey. This method was intended forresearch purposes only and was not subjected to method validation.Samples were analyzed in a 96-well format and quantification wasachieved by comparison of measured fluorescence in the sample to astandard curve. An Odyssey CLx near infrared scanner (Li-CorBiosciences) was used to measure signal from samples and standards,using the 800 nm channel (785 nm excitation). The studies in which thismethod was used are listed in Table 42.

TABLE 42 Listing of Studies for the Analytical Method of Quantitation ofCompound 16 Lowest Species (Method of Study Standard UsedAdministration) Reference Number (ng/mL) CD-1 mouse (IV) ResearchRPT0030 v02 14.6 beagle dog (IV) Xenometrics 10803 0.5 cynomolgus monkey(IV) Xenometrics 10822 0.5

LC/MS methods were developed and used to analyze serum from a non-GLPrat study and from GLP studies in rats and cynomolgus monkeys. The LC/MSmethods were validated prior to GLP study sample analyses. The LowestLevel of Quantitation (LLOQ) for Compound 16 was 10 ng/mL and 5ng/mL inrat and monkey serum, respectively. The studies in which these methodswere used are listed in Table 43.

TABLE 43 Listing of Studies for the LC/MS Analysis of Compound 16 inSerum Species LLOQ (Method of Administration) Study Reference Number(ng/mL) Sprague Dawley rat (IV) Xenometrics 10777 10.0 Sprague Dawleyrat (IV) Xenometrics 10865 10.0 cynomolgus monkey (IV) SNBL.814.02 5.0

The stability of rat serum samples stored at −70° C. was at least 9months. Stability data from the monkey support serum sample storage upto 1 month at −70° C. Incurred sample reanalysis (ISR) was performed onsamples from the rat and monkey GLP studies. The rat ISR assessmentpassed, however the monkey ISR did not. Follow-up investigations of theISR failure in the monkey study were conducted in an attempt to identifya root cause and assess impact of the ISR failure on the study data. Nosingle root cause was identified, however issues with sample stabilityand variability from the various lots of control serum and matrix likelycontributed. It was noted that the failure rate was highest in sampleswith relatively low concentrations of Compound 16.

Analytical Method for Quantitation of Compound 16 in Humans. Based onthe methods used to detect Compound 16 in rat and monkey serum, an LC/MSassay to detect Compound 16 in human serum was developed. This methoddiffers from the validated preclinical methods used in rat and monkey ina few aspects. First, the internal standard used in the human method isan isotope-labeled version of Compound 16 which is approximately 30Daheavier than the preclinical standard, Compound 76 (the peptidecomponent of Compound 76 having a sequence ofH-Met-Cys-Met-Pro-Cys-Phe-Thr-Thr-Asp-His-Gln-Met-Ala-Arg-Ala-Cys-Asp-Asp-Cys-Cys-Gly-Gly-Ala-Gly-Arg-Gly-Lys-Cys-Tyr-Gly-Pro-Gln-Cys-Leu-Cys-Arg-OH).Second, bovine serum albumin has been added to the standards and qualitycontrol solutions to improve stability. Third, an AB Sciex QTrap 5500mass spectrometer was used in place of the Sciex API 5000. The LLOQ forthe human assay is 10 ng/mL.

Pharmacokinetic Methods. Compound 16 serum concentration versus timedata were downloaded into Phoenix WinNonlin 6.3 (Pharsight, Cary, N.C.)for analyses using standard noncompartmental methods of intravenousbolus, intravenous infusion, or extravascular input as appropriate.Mouse and rat PK data were analyzed using the mean serum concentrationversus time data. Dog and monkey PK data were analyzed by individualanimal and then group summary statistics were calculated. Samples thatwere not analyzed for Compound 16 concentration were due to insufficientsample volume or concentration values below the limit of quantitation ofthe assay. Such samples were treated as missing for the purpose ofcalculating toxicokinetic (TK) parameters, and were not included in thecalculation of group means. Nominal dose and sample collection timeswere used in estimating parameters.

Absorption. Compound 16 was administered IV as the intended clinicalroute of administration is IV.

Distribution:

Pharmacokinetics of Intravenous Administration of Compound 16 in FemaleCD-1 Mice. Female mice were intravenously or subcutaneously administereda single fixed dose of 0.02 mg of Compound 16. Compound 16 wasformulated in 10 mM Tris/5% dextrose. Serum was collected from mice at0.25, 2, 6 and 24 hours following administration (FIG. 22 ). Afluorescence-based method was used to measure Compound 16 serumconcentrations.

Tolerability and Toxicokinetic Study of Compound 16 in Male SpragueDawley Rats. Toxicokinetics (TK) were evaluated in male Sprague Dawleyrats as part of a single dose tolerability study. Rats wereintravenously administered a single fixed bolus dose of Compound 16 atone of two nominal dose levels, either 0.03 or 0.3 mg. Compound 16 wasformulated in 10 mM Tris, 5% Mannitol, pH 7.2. Animals (n=3 pertimepoint) were bled on a staggered sampling scheme at 0.083, 0.33, 1,2, 4, 8, 24, and 48 hours after injection. An LC/MS method was used tomeasure Compound 16 serum concentrations.

Following a single IV bolus, serum concentrations were measurable up to4 hours in the 0.03 mg group and up to 8 hours in the 0.3 mg group (FIG.23 ). Compound 16 exposure based on C₀ and AUC_(0-t) increased in anapproximately dose-proportional manner. Due to the limited measurableconcentrations available in the PK profile, parameters based on theterminal phase could not be calculated.

Biodistribution was evaluated by measuring fluorescence intensity intissue sections from the organs collected at euthanasia 48 hours afterintravenous administration. Kidney, liver, heart, and aorta werecollected and fixed in 10% formalin. Tissue was stored at 4° C. in 10%formalin until processed. The tissue was washed twice in PBS thenprocessed through a sucrose gradient of 10% sucrose/PBS for 5 hoursfollowed by 20% sucrose/PBS overnight at 4° C. for cryoprotection. Thetissue was then gross sectioned and frozen in OCT on dry ice. The frozentissue blocks were sliced into 12 μm sections and placed on gelatincoated slides. Slides were scanned using the Odyssey CLx imaging system(Li-Cor Biosciences) using the 800 nm channel (785 nm excitation).Images were analyzed using Image Studio software (Li-Cor Biosciences) bymeasuring the signal within a region of interest (ROI) for each sample.An average signal for three to four sections for each animal wascalculated.

The signal emitted from Compound 16 was highest in the kidney which wasconsistent with renal clearance of the product. Less intensefluorescence was observed in liver and aorta. Signal was low in theheart. Signal increased with dose escalation in all of the tissues thatwere tested; however, the signal in the heart remained low even at highdoses. Signal in the aorta and the great vessels of the heart wasrelatively high compared to the heart and increased with doseescalation. Distribution of Compound 16 to the kidney, liver, heart, andaorta did not seem to have toxicological significance. There were nocorresponding changes in serum chemistry or histopathology.

Pilot Pharmacokinetics of Compound 16 Following IntravenousAdministration to Male Beagle Dogs. Two male beagle dogs wereintravenously administered a nominal dose of 1 mg of Compound 16. Thefirst dog received the dose as an IV bolus and the second dog receivedthe dose as a 15 minute IV infusion. Compound 16 was formulated in 10 mMTris, 5% Mannitol, pH 7.2. Animals were bled for PK analysis atpre-dose, 0.083, 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, and 96 hourpost-dose (bolus) or post-start of infusion. A fluorescence-based methodwas used to measure Compound 16 serum concentrations. (Table 44).

Following a single IV bolus to Dog X, the C₀ value was consistent withan administered dose of 0.36 mg (0.36 mg/515 mL plasma volume in a dog.The tl/2 was approximately 54 hours, CL was 1100 mL/hr, and V_(ss l was)29900 mL which was approximately twice total body water (Davies andMorris 1993). (Table 44).

TABLE 44 Serum concentrations of Compound 16 (ng/ml) following a singleintravenous dose (bolus or infusion) of 1 mg to male dogs, calculated bycomparison to standard curve. Dog X2 (15- Time Dog X1 (IV min IV (hr)bolus) infusion) 0 BLQ BLQ 0.083 391.89 54.61 0.25 209.5 225.74 0.573.74 36.84 1 68.24 22.89 2 10.43 6.18 4 2.63 1.61 6 1.58 1.13 24 1.320.56 48 0.81 0.32 72 0.52 0.21 96 0.44 0.18 BLQ = below limit ofquantification. Time = 0 is pre-dose.

Following a 15 minute IV infusion to Dog X², the t_(1/2) at 57 hours wasconsistent with that of Dog X1. However, Cmax was approximately 40%lower relative to the IV bolus C_(max). Overall exposure based on AUCfollowing the infusion was approximately 55% lower than that observedfollowing the IV bolus dose. This corresponded to faster CL and higherV_(ss) values relative to the IV bolus dose. It is unknown if thisobserved difference in CL and V_(ss) is due to a true PK differencebetween the animals or dosing routes, or an artifact of issues with thedosing solution and a potentially incomplete priming of the infusionline, leading to a lower than expected administered dose. (Table 44).

Pilot Pharmacokinetics of Compound 16 Following IV Administration toMale Non-Human Primates. Two cynomolgus monkeys were administered anominal dose of 0.6 mg of Compound 16 as an IV bolus. Compound 16 wasformulated in 10 mM Tris, 5% Mannitol, pH 7.2. Animals were bled for PKanalysis at predose, 0.083, 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, and 96hours postdose. A fluorescence-based method was used to measure Compound16 serum concentrations (FIG. 24 ).

The shape of monkey NHP1's serum concentration versus time profile wasnot what would be expected following a single IV bolus dose (Table 44).The reason is unknown and these data could not be used to calculate PKparameters that require definition of the terminal phase of theconcentration versus time profile. The t_(1/2) for NHP2 wasapproximately 55 hours, CL was 170 mL/hr, and V_(ss) was 6580 mL.

Single Dose Intravenous Toxicity and Toxicokinetic Study of Compound 16in Sprague Dawley Rats. Male and female Sprague Dawley rats wereadministered a single IV bolus dose of Compound 16 at fixed doses of0.07, 0.7, and 7 mg. Compound 16 was formulated in 10 mM Tris, 5%Mannitol, pH 7.2. PK samples were obtained using a staggered samplingscheme of 3 males and 3 females per timepoint per group at pre-dose,0.25, 1, 3, 6, 12, 24, and 48 hours post-dose (FIG. 25 ). Serum sampleswere analyzed for Compound 16 concentration via a validated LC/MS basedprocedure and the resulting concentration versus time data were used toestimate TK parameters using non-compartmental analysis.

Following a single fixed IV bolus dose of Compound 16, mean serumconcentrations were measurable out to 12 hours post-dose with an LLOQ of10 ng/mL in the 0.07 and 0.7 mg dose groups and out to 48 hourspost-dose in the 7 mg dose group. Exposure based on C_(max) and C₀increased in an approximately dose-proportional manner across the testeddose range. AUC_(0-t) was approximately dose-proportional between the0.07 and 0.7 mg dose levels, and increased in a higher thandose-proportional manner between the lower dose groups versus the 7 mgdose level (see Table 45). Although C_(max) or C₀ values were variablebetween males and females, there was no obvious overall trend based onthese parameters that would indicate an effect of sex. AUC_(0-t) wasconsistent between males and females across all dose groups.

TABLE 45 Mean non-compartmental Compound 16 PK Parameters Following aSingle IV Bolus Dose to Male and Female Sprague Dawley Rats. Note: Dueto the unequal number of samples with measurable Compound 16concentrations between males and females at certain timepoints, someoverall dose group (male +female) exposure parameters do not equal theaverage of the corresponding male and female exposure values. DoseC_(max) C₀ AUC_(0-t) (mg) Sex (ng/mL) (ng/mL) (hr*ng/mL) 0.07 Male 7891500 821 Female 541 921 700 Male + Female 665 1200 794 0.7 Male 813016400 8130 Female 6880 11000 7850 Male + Female 7510 13400 8070 7 Male83300 117000 127000 Female 95400 155000 134000 Male + Female 89400136000 130000

A Single Dose 14-Day Intravenous Toxicity Study of Compound 16 inCynomolgus Monkeys. Male and female cynomolgus monkeys (n=3 males and 3females per group) were administered a single IV bolus dose of Compound16 at fixed doses of 0.6, 6, and 60 mg. Compound 16 was formulated in 10mM Tris, 5% Mannitol, pH 7.2. PK samples were obtained pre-dose, 0.083,0.25, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, and 120 hours post-dose. Serumsamples were analyzed for Compound 16 concentration using a validatedLC/MS based procedure and the resulting concentration versus time datawere used to estimate TK parameters using non-compartmental analysis.

Mean serum concentrations of greater than 5 ng/mL were measured at 8,48, and 120 hours post-dose in the 0.6, 6, and 60 mg dose groups,respectively (FIG. 26 ). Exposure based on Co and AUC_(0-t) was 6 to 29%higher in females relative to males across the tested dose levels (seeTable 46). This is potentially due to the female monkeys' smaller sizerelative to the male monkeys since Compound 16 was administered as afixed dose, rather than based on body weight. However, alldose-dependent trends in exposure were consistent between the sexes.

TABLE 46 Mean Non-compartmental Compound 16 PK Parameters Following aSingle IV Bolus Dose to Male and Female Cynomolgus Monkeys. Dose C_(max)C₀ AUC_(0-t) t½ CL Vss (mg) Sex (ng/mL) (ng/mL) (hr*ng/mL) (hr) (mL/hr)(mL) 0.6 Male 3930 4760 3310 ND ND ND Female 4950 5770 4280 ND ND NDMale + Female 4440 5260 3800 ND ND ND 6 Male 55400 67700 85800 ND ND NDFemale 63700 78700 90000 ND ND ND Male + Female 60300 74300 88300 ND NDND 60 Male 414000 433000 1120000 36.8 54.5 230 Female 457000 4600001360000 30.6 46.7 192 Male + Female 436000 447000 1240000 33.7 50.6 211ND: not determinable due to limited concentration versus time data inthe terminal phase.

Exposure based on C_(max) and C₀ generally increased in adose-proportional manner, although the 6 mg dose level had higher thanexpected C_(max) and C₀ values. Exposure based on AUC_(0-t) valuesincreased in a greater than dose-proportional manner across all dosegroups, suggesting that Compound 16 clearance is reduced at higher doses(see Table 46). This also might be due, in part, to an incompletecharacterization of the TK profile at the lowest dose level due to assaylimitations. There also appeared to be a slower rate of decline inCompound 16 concentrations between 0.25 and 4 hours post-dose in the 6mg dose group relative to the 0.6 mg dose group.

Additional non-compartmental TK parameters were able to be estimated forthe 60 mg dose group. There were no substantial differences in the TKparameters between males and females. The overall mean t_(1/2), CL, andV_(ss l were) 33.7 hour, 50.6 mL/hr, and 211 mL, respectively.

Metabolism (Interspecies Comparison). No metabolism studies have beenconducted with Compound 16. The ICG portion of the molecule is known tobe mainly excreted unchanged into the bile, and does not undergo anyappreciable metabolism.

Excretion. Urine excretion has not been formally assessed. Based onbiodistribution data in select normal tissues from single dose mousetumor models and a non-GLP single dose rat study, the kidney appears tobe an important organ involved in the clearance and elimination ofCompound 16. This is further supported by data from the single-dose GLPrat study in which green discolored kidneys were noted in the highestdose group 2 days after injection, and the appearance of green coloredurine in the single-dose GLP monkey study.

Pharmacokinetic Drug Interaction. No drug interaction studies have beenconducted with Compound 16.

Compound 16 PK profiles following IV administration demonstrated abi-exponential decline with a rapid initial phase and a longer terminalphase in all species. Based on PK data from the rat and monkeysingle-dose GLP studies, Compound 16 exposure based on C₀ or C_(max)values appeared to be approximately dose proportional over the testeddose ranges. In contrast, AUC values increased in a dose-proportional orhigher than dose-proportional manner, suggesting that Compound 16clearance is reduced at higher doses. The terminal phase could becharacterized in the high dose group (60 mg) in cynomolgus monkeys,allowing estimation of t_(1/2), CL, and V_(ss) parameters. The meant_(1/2), CL, and V_(ss) values were 33.7 hours, 50.6 mL/hr, and 211 mL,respectively.

There was no obvious effect of gender on Compound 16 PK in the rat, butexposure was consistently higher in female monkeys compared to malemonkeys. This finding is perhaps due to the size difference in males andfemales since Compound 16 was administered at fixed dose levels.

Example 23 Pharmacokinetics of Other Chlorotoxin Conjugate Compounds

This example demonstrates the pharmacokinetic (PK) profile of Compounds1-720 following a single intravenous (IV) injection in mice, rats, dogs,and monkeys.

Materials and methods are as described in Example 22 but with Compounds1-720.

Pilot Pharmacokinetics Study of Compounds 1-720 Following IVAdministration to Male Non-Human Primates (Cynomolgus). Cynomolgusmonkeys are intravenously administered a single bolus injection ofCompounds 1-720 at a fixed dose level. Clinical observations are made ateach time point.

In the pilot studies, pharmocokinetic (PK) profiles of Compounds 1-720following IV administration demonstrate a bi-exponential decline with arapid initial phase and a longer terminal phase in all species.

Following IV administration of Compounds 1-720 in the rat and monkeysingle-dose GLP toxicology studies, exposure based on C_(max) and C₀increases in an approximately dose-proportional manner across the testedranges. AUC_(0-t) values increase in a dose-proportional or higher thandose-proportional manner. These observations suggest that conjugateCompounds 1 to 720_clearance is reduced at higher doses.

Pharmacokinetics of Intravenous Administration of Compounds 1-720. Inseparate experiments, mice, rats, dogs and monkeys are intravenouslyadministered as an IV bolus or IV infusion of Compounds 1-720. Serum iscollected at multiple time points following administration. Afluorescence-based method is used to measure Compounds 1-720 serumconcentrations. C_(max), C₀, t_(1/2) and AUC_(0-t) values are obtainedfrom the resulting data. Biodistribution is evaluated by measuringfluorescence intensity in tissue sections from the organs collected ateuthanasia at time-points following intravenous administration.Distribution of Compounds 1-720 to the organs typically does not seem tohave toxicological significance, with no corresponding changes in serumchemistry or histopathology.

Example 24 Pharmacokinetics of Compound 16 in Humans

This example demonstrates the pharmacokinetics of Compound 16 in humansubjects with nonmelanotic skin cancer. The primary objective of thestudy was to evaluate the safety and tolerability of a single IVadministration of Compound 16.

At the time of filing, the study was ongoing. Interim data from thefirst cohorts evaluated are summarized in this example.

Following a single IV administration, the maximum serum Compound 16concentration was observed at the end of the infusion. Drug levels weredetectable hours after infusion. Exposure based on C_(max) and AUC_(0-t)increased in a dose-dependent manner. The results indicate thatpharmacokinetic data obtained using animal models is predictive ofpharmacokinetics in human patients.

Overall, single IV administrations of Compound 16 were well tolerated.No significant or clear pattern of toxicities has been observed.

Example 25 Pharmacokinetics of Chlorotoxin Conjugate Compounds in Humans

This example demonstrates the pharmacokinetics of a chlorotoxinconjugate in human subjects.

Study Design:

Subjects are given intravenous (IV) bolus injections of 1 mg, 3 mg, 12mg or 30 mg of a chlorotoxin conjugate such as Compound 16. Bloodsamples are collected before injection (time=0 hours) and at 30, 60, 90,120, 180 and 240 minutes post-injection. Samples are analyzed withfluoresence-based methods and with liquid chromatography/massspectrometry (LC/MS) method to determine pharmacokinetic profiles ofchlorotoxin conjugate in humans (FIG. 27 ).

Initial blood serum concentration and area under the curve data areconsistent with predictions from animal models (Table 47, Table 48).

TABLE 47 Initial blood serum concentration (C₀) and area under curve(AUC) percentile values at different time points following a 1 mg IVbolus dosing of Compound 16 into human patients. Parameter Estimate C₀172.00 ng/mL    AUC_(Last) 85.27 hr*ng/mL AUC₂₅ 21.37 hr*ng/mL AUC₅₀42.67 hr*ng/mL AUC₇₅ 64.07 hr*ng/mL

TABLE 48 Times when different area under curve (AUC) percentiles arereached following a 1 mg IV bolus dosing of Compound 16 into humanpatients. Start Time End Time Time (hr) (hr) Label (mm) 0 0.143 AUC258.6 ± 5.0 0 0.344 AUC50 20.6 ± 7.8  0 0.690 AUC75 41.4 ± 15.1

Example 26 Intraoperative Imaging and Tumor Identification

This example describes the tumor-binding specificity of BLZ-100 and theratio between tumor and background binding by BLZ-100.

Intraoperative Imaging

A prototype intraoperative imaging system was utilized for patients 17through 28 from the study described in Example 9. Its use duringsurgeries enabled imaging of tumor beds as well as tumors in situ andimmediately following excision. The data show generally gooddiscrimination between gross tumor and surrounding tissues. Peritumoralskin tended to have background fluorescence, while uninvolved skin hadlower fluorescence. Mucosal tissues also showed background fluorescence,resulting in lack of specificity and residual non-tumor fluorescence inpatient 17. Tumor bed imaging showed little or no background staining in“internal” tissues such as trachea, muscle, and fat. The intraoperativeimaging showed good subjective concordance with the quantitative ex vivoimage analysis conducted using the Odyssey scanner. The tumors that hadoverall high intensity and good tumor to normal ratios ex vivo alsoshowed high contrast and were easy to detect intraoperatively (Table49).

TABLE 49 Summary of intraoperative imaging observations. Imaging was notperformed on patient 25 due to technical issues with the instrument. TBRwas calculated when both tumor and normal tissue were imagedsimultaneously. TBR Delineation of Residual Clean (gross, TBR tumorfluorescence in surgical Patient Tumor type ex vivo) (Intraoperative)during surgery tumor bed margins 17 Fibrosarcoma <1 <1 no yes yes 18Mastocytoma 1.5 1.5 yes no yes 19 Soft tissue sarcoma 5 19 yes no yes 20Follicular 2 ND yes no yes carcinoma 21 Adenocarcinoma 3 ND yes no no-invasion into vessels 22 Adenocarcinoma 2.5 5 yes, multiple no yeslesions 23 Squamous cell 5 8 yes no yes carcinoma 24 Adenocarcinoma 2 3yes no yes 26 Soft tissue sarcoma 2 2 yes, multiple yes no lesions newand previously irradiated 27 Meningioma 2.5 2.5 yes yes no 28Hemangiosarcoma <1 <1 no yes no ND = not determined.

Several cases are presented as examples of the clinical utility of achlorotoxin conjugate with intraoperative imaging.

Patient 19, Soft-Tissue Sarcoma

Patient 19 had a grade II soft-tissue sarcoma on her foreleg. The tumorhad been clinically evident for several months without treatment. Theperitumoral skin was swollen and ulcerated (FIG. 28 , panel A).Intraoperative imaging of the tumor in situ showed variable fluorescencein the tumor, some fluorescence in the swollen and ulcerated peritumoralskin, and little or no background fluorescence in other areas (FIG. 28 ,panels B and C). Imaging of the tumor immediately post excision showedroughly 8-fold variability of fluorescence intensity within the tumor(FIG. 28 , panels D and E). The variation within the tumor is consistentwith the pathology report that approximately 50% of the mass is replacedby eosinophilic debris (necrosis). Imaging of the tumor bed showedfluorescence in peritumoral skin; a sample of this skin was resected andsent for further imaging and histopathology. There was no residualfluorescence in the tumor bed or in the surrounding uninvolved skin(FIG. 28 , panel F). A sample of resected peritumoral skin was sent forfurther imaging and histopathology, which confirmed the absence ofneoplastic invasion.

Patient 22, Mammary Carcinoma

Patient 22 had a recurrent mammary adenocarcinoma. The tumor was removeden bloc with overlying skin and surrounding fatty tissue. Imaging of thetumor from the bottom showed that the mass is detectable through ˜0.5 cmof normal fatty tissue (FIG. 29 , panel A). The diffuse appearance ofthe fluorescence is due to tissue scattering of the emitted light. Aslice for further imaging was removed from the skin side, leaving thebulk of the mass and surrounding tissue exposed. The contrast betweenthe tumor and surrounding tissue is improved due to the absence ofintervening tissue (FIG. 29 , panels B and C).

The tissue pieces collected for further imaging contained gross tumor(white areas, FIG. 29 , panel D) and adjacent tissue. Fluorescenceimaging shows about 2.5-fold brighter fluorescence in the gross tumorareas compared with the adjacent tissue (FIG. 29 , panel E). The tumorbed showed no residual fluorescence (FIG. 29 , panel F). Note that thefluorescence in the skin appears brighter in panel F than in panel C;this is due to the increased sensitivity used in the survey of the tumorbed, to ensure that any residual fluorescence would be detected.

Patient 23, Cutaneous Squamous Cell Carcinoma

Patient 23 had a cutaneous squamous cell carcinoma of the tail. Thelesion had penetrated the skin, which was grossly swollen and ulcerated(FIG. 30 , panel A). The lesion was covered by a serocellular crust.Preoperative fluorescence imaging showed very little fluorescencepenetrating the serocellular crust, while the peritumoral skin showedrelatively bright staining (FIG. 30 , panel B). Two “fingers” offluorescence were noted, which extended to the opposite side of the tail(FIG. 30 , panel C).

Following removal of the tail, tissues were imaged and sections wereremoved for further imaging. A section of central tumor (FIG. 30 ,panels D and E, at right) showed relatively intense fluorescence. Theremaining central tumor, viewed from the side rather than through theserocellular crust, showed fluorescence intensity similar to that of thecentral tumor. The peritumoral skin was less intense than the tumoritself (FIG. 30 , panel F), but was about 3-fold more intense thanuninvolved skin. Samples of skin from the fluorescent areas on theopposite side of the tumor were submitted for histopathology, and theydid not contain tumor.

Patient 20, Thyroid Carcinoma

Patient 20 had a thyroid carcinoma. The entire thyroid gland wasremoved, along with an enlarged lymph node. Intraoperative imaging ofthe thyroid showed most of the gland was fluorescent, with about 2-foldvariation in signal intensity throughout (FIG. 31 ). The lymph node hadregions of fluorescence that were comparable to the primary tumor. Thetumor bed had no residual fluorescence. Additionally, no significantnon-specific background fluorescence was observed in the internalstructures including the trachea, nerves, and arteries. Histopathologyshowed that the thyroid was 95% effaced by the tumor, which had largeareas of blood filled spaces and necrosis. These may account for thevariability in staining seen in the primary tumor. The lymph node wasconfirmed to contain metastatic disease.

Example 27 Specificity and Tumor to Background Ratio of OtherChlorotoxin Conjugate Compounds

This example describes the tumor-binding specificity of Compounds 1-720and the ratio between tumor and background binding by Compounds 1-720.

Intraoperative Imaging

Methods and materials used are as described in Example 26, but withCompounds 1-720. A prototype intraoperative imaging system is utilized.Its use during surgeries enables imaging of tumor beds as well as tumorsin situ and immediately following excision. The data show generally gooddiscrimination between gross tumor and surrounding tissues. Peritumoralskin tends to have background fluorescence, while uninvolved skin haslower fluorescence. Tumor bed imaging shows little or no backgroundstaining in “internal” tissues such as trachea, muscle, and fat. Theintraoperative imaging shows good subjective concordance with thequantitative ex vivo image analysis conducted using the Odyssey scanner.The tumors that have overall high intensity and good tumor to normalratios ex vivo also show high contrast and are easy to detectintraoperatively.

Example 28 Histopathologic Scoring, Sensitivity and Specificity

This example describes the evaluation of tumor and adjacent tissues atthe cellular level in order to assess sensitivity and specificity of achlorotoxin conjugate, such as BLZ-100 for cancer cells.

Evaluation of canine tumor and adjacent tissues at the cellular levelwas performed in order to assess sensitivity and specificity of BLZ-100for cancer cells. For this analysis, two cutaneous squamous cellcarcinomas, three mammary cancers, and four subcutaneous soft tissuesarcomas were included. Sensitivity and specificity was calculated usinga grid analysis on 30 micron frozen sections. An overlay of eachfluorescence image with the corresponding H&E stained image that wasscored as tumor or normal by a histopathologist was analyzed. Thisanalysis was performed separately for each case.

The data were grouped by individual section and plotted for eachpatient. The subcutaneous soft tissue sarcomas showed highly specifictumor fluorescence. A logistic regression analysis was used to determinea reasonable threshold intensity for detecting tumor in the subcutaneoussoft-tissue sarcomas. Non-skin tissues were used to compute sensitivityand specificity, with a threshold intensity of 30,000 used as a cutoffvalue. Grid squares were called tumor or no tumor based on fluorescenceintensity and based on pathologist call. Concordant and discordant callsare used to calculate sensitivity and specificity. Sensitivity (95%) andspecificity (85%) were very good using a threshold grid squarefluorescence of 30,000. Peritumoral skin in patient 19 was above thisthreshold in all grid squares; as discussed in Example 26, this patienthad an ulcerated tumor and grossly edematous skin immediately adjacentto the mass. The elevated signal in this patient's skin sample accountedfor all above-threshold data points in this analysis.

The cutaneous squamous cell carcinomas had fluorescence signal comingboth from the tumor and from the underlying dermis. In most cases, thesignal was brighter in the underlying dermis, leading to the “inverted”tumor vs. normal intensity. Although histologic specificity in thesetumors is low, truly uninvolved skin seen during intraoperative imagingin patient 23 and during ex vivo imaging in patient 14 was notfluorescent. Analysis of the mammary tumors shows that, like skin,mammary tissue adjacent to tumor takes up BLZ-100. In both tumor types,gross tumors had higher fluorescence than uninvolved tissue.

For this analysis, two cutaneous squamous cell carcinomas, three mammarycancers, and three subcutaneous soft-tissue sarcomas were included.Tissues were sectioned on a cryostat, and 30 micron sections were imagedon the Odyssey scanner. These sections or serial sections were stainedwith H&E and read by an expert histopathologist who was blinded to thefluorescence data. A grid was overlaid on the fluorescence image, andtotal fluorescence in each grid square was measured using Image Studio(Li-Cor) software provided with the Odyssey scanner. Overlay of thefluorescence image with the scored H&E image enabled calling of tumorvs. non-tumor for each grid square.

For each tumor, grid analysis was done on sections from different areasof tumor and adjacent non-tumor tissue, as well as samples of uninvolvedtissue when available. As discussed above, background staining in theswollen, ulcerated skin in patient 19 caused suspicion of tumorinfiltration during the surgery. The fluorescence analysis in sectionsof this skin would lead to the same conclusion. Note that the uninvolvedskin samples from patients 12 and 13 are well below the threshold forbeing incorrectly identified as tumor tissue.

Example 29 Histopathologic Scoring, Sensitivity and Specificity ofChlorotoxin Conjugate Compounds

This example describes the evaluation of tumor and adjacent tissues atthe cellular level in order to assess sensitivity and specificity of achlorotoxin conjugate, such as Compound 16 for cancer cells.

Materials and methods used were as described in Example 28. FIG. 32shows box & whiskers plots of fluorescence intensity in grid squaresfrom multiple patients and for each tissue section analyzed (T, tumor.NT, adjacent non-tumor tissue. PS, peritumoral skin. S, uninvolvedskin). Sensitivity (95%) and specificity (85%) were very good using athreshold of 30,000 (arbitrary units). Peritumoral skin in patient 19was above this threshold in all grid squares. This patient had anulcerated tumor and grossly edematous skin immediately adjacent to themass. The elevated signal in this patient's skin sample accounted forall above-threshold data points in this analysis.

Grid squares were called tumor or no tumor based on fluorescenceintensity and based on pathologist call. Concordant and discordant callsare used to calculate sensitivity and specificity. The results are shownin Table 50.

TABLE 50 Sensitivity and specificity analysis for subcutaneoussoft-tissue sarcoma samples. Tumor No Tumor (pathologist) (pathologist)Total Tumor (intensity) 111 0 111 No Tumor (intensity) 7 11 18 Total 11811 129 Kappa coefficient (95% CI): 0.730 (0.543, 0.917); Sensitivity(95% CI): 94.1% (88.2%, 97.6%); Specificity (95% CI): 100.0% (71.5%,100.0%).

Example 30 Histopathologic Scoring, Sensitivity and Specificity of OtherChlorotoxin Conjugate Compounds

This example describes the evaluation of tumor and adjacent tissues atthe cellular level in order to assess sensitivity and specificity of achlorotoxin conjugate, such as Compounds 1-720 for cancer cells.

Evaluation of canine tumor and adjacent tissues at the cellular level isperformed in order to assess sensitivity and specificity of Compounds1-720 for cancer cells. Sensitivity and specificity are calculated usinga grid analysis on 30 micron frozen sections. An overlay of eachfluorescence image with the corresponding H&E stained image that wasscored as tumor or normal by a histopathologist is analyzed. Thisanalysis is performed separately for each case. Cutaneous squamous cellcarcinomas, mammary cancers, and subcutaneous soft-tissue sarcomas areevaluated. Tumor tissue and tissue adjacent to tumors has higher meanfluorescence than does normal tissue.

Example 31 Labeling of Additional Types of Tumors

This example describes the use of BLZ-100 for the labeling of othermiscellaneous tumor types not described in the preceding examples.Besides the above-described tumor types, there were several tumor typesfor which the number of patients was insufficient to conduct meaningfulsensitivity and specificity analysis. These include mast cell tumors(N=1 at effective dose), lung cancer (N=1), and meningioma (N=1). Therewere two thyroid carcinomas in which signal in 30 micron sections wastoo low to permit the analysis. The oral tumors were determined to benon-specific on gross imaging.

Lung cancer is of potential interest clinically. The results of grossimaging in the canine lung cancer suggest specific tumor uptake, with3:1 TBR compared with adjacent lung or with uninvolved skin. There was asmall suspected metastasis seen in the adjacent lung tissue. Onhistopathologic analysis, the signal intensity was low but measurable,and was specific for tumor in the primary mass. The suspected metastasiscould not be confirmed due to frozen section artifact.

Brain tumors are of very high interest for clinical and commercialdevelopment of a chlorotoxin conjugate. There was one brain tumor caseenrolled in the study, a meningioma. Meningiomas are extra-axial tumorswith typically low histologic grade in dogs as well as in humans.Intraoperative imaging showed signal in the chlorotoxinconjugate-labeled tumor with a 2.5-fold tumor to background (normalbrain) ratio. The surgical approach was through the sinus, so nasalmucosa was present in the image. The mucosal tissues are a known sourceof background, and in this case provided a positive control forfluorescence intensity. Tumor was removed in fragments; pieces to beanalyzed were embedded in OCT and snap-frozen. Imaging of 30 micronsections showed low but detectable signal. The optimal dose for CNStumors arising within the blood-brain barrier will have to be determinedwith additional subjects, including cases with malignant tumors such asglioma. However, this case did provide an opportunity for imaging ofnormal brain tissue, and it demonstrated that a low-grade brain tumorcan be successfully imaged. This is significant, since completeresection in low-grade tumors can be curative.

Example 32 Labeling of Additional Types of Tumors Using ChlorotoxinConjugate Compounds

This example describes the use of Compound 16 for the labeling of othertumor types.

Intraoperative imaging of a canine meningioma showed signal in theCompound 16-labeled tumor (FIG. 33 , panel A), with a 2.5-fold tumor tobackground (normal brain) ratio. The surgical approach was through thesinus, so nasal mucosa was present in the image. The mucosal tissues area known source of background, and in this case provided a positivecontrol for fluorescence intensity. Tumor was removed in fragments;pieces to be analyzed were embedded in OCT and snap-frozen. Imaging of30 micron sections showed low but detectable signal (FIG. 33 , panels Band C). H&E stained sections are shown for comparison (FIG. 33 , panelsD and E). This case demonstrated that a low-grade brain tumor can besuccessfully imaged.

Example 33 Labeling of Additional Types of Tumors Using OtherChlorotoxin Conjugate Compounds

This example describes the use of Compounds 1-720 for the labeling ofmiscellaneous tumor types, such as mast cell tumors, lung cancer,meningioma, thyroid carcinomas, and oral tumors.

Intraoperative tumor imaging shows signals emitted by Compounds 1-720.The tumors are removed in fragments; pieces to be analyzed are embeddedin OCT and snap-frozen. Imaging of 30 micron sections shows low butdetectable signal. H&E stained sections are used for comparison. Tumorsare successfully identified using Compounds 1-720 and optimal doses aredetermined.

Example 34 Manufacture of Peptides

The peptide sequence was reverse-translated into DNA, synthesized, andcloned in-frame with siderocalin using standard molecular biologytechniques. (M. R. Green, Joseph Sambrook. Molecular Cloning. 2012 ColdSpring Harbor Press.). The resulting construct was packaged into alentivirus, transfected into HEK293 cells, expanded, isolated byimmobilized metal affinity chromatography (IMAC), cleaved with tobaccoetch virus protease, and purified to homogeneity by reverse-phasechromatography. Following purification, each peptide was lyophilized andstored frozen.

Example 35 Radiolabeling of Peptide

This example describes radiolabeling of peptides with standardtechniques. See J Biol Chem. 254(11):4359-65 (1979). The sequences wereengineered to have the amino acids, “G” and “S” at the N terminus. SeeMethods in Enzymology V91:1983 p. 570 and Journal of BiologicalChemistry 254(11):1979 p. 4359. An excess of formaldehyde was used toensure complete methylation (dimethylation of every free amine). Thelabeled peptides were isolated via solid-phase extraction on Strata-Xcolumns (Phenomenex 8B-S100-AAK), rinsed with water with 5% methanol,and recovered in methanol with 2% formic acid. Solvent was subsequentlyremoved in a blowdown evaporator with gentle heat and a stream ofnitrogen gas.

Example 36 Peptide Detectable Agent Conjugates

This example describes the dye labeling of peptides. A peptide of thedisclosure is expressed recombinantly or chemically synthesized, andthen the N-terminus of the peptide is conjugated to a detectable agentvia an NHS ester using DCC or EDC to produce a peptide-detectable agentconjugate. The detectable agent is the fluorophore dye is a cyanine dye,such as Cy5.5 or an Alexa fluorophore, such as Alexa647.

The peptide detectable agent conjugates are administered to a subject.The subject can be a human or a non-human animal. After administration,the peptide detectable agent conjugates home to cartilage. The subject,or a biopsy from the subject, can be imaged to visualize localization ofthe peptide detectable agent conjugates to cartilage. In some aspects,visualization of the peptide detectable agent conjugates in cartilageafter administration results in diagnosis of arthritis, cartilagedamage, or any cartilage disorder.

Example 37 Method to Determine Improved Peptide Variants

This example shows a method for determining ways to improve peptidevariants by comparing and analyzing the primary sequences and tertiarystructures of scaffold peptides. FIG. 38A-FIG. 38C show sequences of SEQID NO: 1025 aligned with SEQ ID NO: 800, SEQ ID NO: 1025 aligned withSEQ ID NO: 1026, and SEQ ID NO: 1025 aligned with SEQ ID NO: 967. Thesequence alignment of the two scaffolds was used to identify conservedpositively charged residues (shown in boxes) that may be important forcartilage homing. A peptide of SEQ ID NO: 967 homes to cartilage andother peptides with positively charged residues in similar positions, orcysteines in similar positions, or other residues that are in similarpositions are also predicted to home to cartilage.

FIG. 39 shows sequences of SEQ ID NO: 804 aligned with SEQ ID NO: 968.The sequence alignment of the two scaffolds was used to identify thebasic/aromatic dyad that may be involved in the interaction with the Kvion channel (K27 and Y36 of SEQ ID NO: 968). The mutation of K27 toalanine, arginine, or glutamic acid destroyed activity against the squidKv1A ion channel K27 and Y36 may be desirable to maintain or add to acartilage homing peptide of this disclosure to maintain or improvehoming, to maintain or improve residence time in cartilage, or tomaintain or improve modulation of an ion channel such as Kv. Incontrast, K27 and Y36 may be desirable to mutate out of a cartilagehoming peptide to reduce interaction with an ion channel such as Kv.Disruption of either the basic or aromatic residue eliminates ionchannel activity. In another example, D amino acids are expected toreduce or eliminate binding.

Example 38 Sequence Alignment to pFam00451:toxin_2 Family to IdentifyCartilage Homing Peptides

This example describes a method for identifying new cartilage homingpeptides by sequence alignment to the pFam00451:toxin_2 structural classfamily. The pFam00451:toxin_2 structural class is a family of peptidesrelated by similarities in sequence identity. FIG. 40 illustratesalignment of peptides within the pfam00451:toxin_2 structural classfamily of SEQ ID NO: 978—SEQ ID NO: 1024. Boxed and bolded residuesindicate relative conservation of sequence while non-boxed andnon-bolded residues indicate areas of higher sequence variability. SEQID NO: 978 was identified as a cartilage homing candidate peptide basedon its structural similarities with the pFam00451:toxin_structural classfamily. FIG. 41 illustrates the sequence alignment of a peptide of SEQID NO: 978 from the pfam00451:toxin 2 structural class family with thesequence of SEQ ID NO: 511. Asterisks indicate positions with a single,fully conserved residue, a colon indicates conservation between groupsof strongly similar properties (scoring >0.5 in the Gonnet pointaccepted mutation (PAM) 250 matrix), and a period indicates conservationbetween groups of weakly similar properties (scoring ≤0.5 in the GonnetPAM 250 matrix). SEQ ID NO: 592 was also identified as a cartilagehoming candidate based on its structural similarities with thepfam00451:toxin 2 structural class family of peptides.

The pFam00451:toxin_2 structural class family is used as a scaffold toidentify variant peptides that have cartilage homing properties. Anymember of the pFam00451:toxin_2 structural class family is used topredict new cartilage homing peptides based on homology, preservedresidues, or a preserved cysteine residue.

Example 39 Dosing of Peptide with Kidney Ligation

This example describes a dosing scheme for administering peptides tomice in conjunction with kidney ligation. Different dosages of thepeptides were administered to Female Harlan athymic nude mice, weighing20g ˜25g, via tail vein injection (n=2 mice per peptide). The sequenceof thirteen cartilage homing peptides of SEQ ID NO: 508—SEQ ID NO: 520are shown in TABLE 51. The experiment was done in duplicates. Thekidneys were ligated to prevent renal filtration of the peptides. Eachpeptide was radiolabeled by methylating lysines and the N-terminus, sothe actual binding agent may contain methyl or dimethyl lysine(s) and amethylated or dimethylated amino terminus.

A target dosage of 50-100 nmol of each peptide carrying 10-25 uCi of ¹⁴Cwas administered to Female Harlan athymic nude mice while anesthetized.Each peptide was allowed to freely circulate within the animal beforethe animals were euthanized and sectioned.

Example 40 Peptide Homing with Kidney Ligation

This example illustrates peptide homing to cartilage of mice withkidneys that were ligated prior to peptide administration. At the end ofthe dosing period in EXAMPLE 39, mice were frozen in a hexane/dry icebath and then frozen in a block of carboxymethylcellulose. Whole animalsagittal slices were prepared that resulted in thin frozen sectionsbeing available for imaging. Thin, frozen sections of animal includingimaging of tissues such as brain, tumor, liver, kidney, lung, heart,spleen, pancreas, muscle, adipose, gall bladder, upper gastrointestinaltract, lower gastrointestinal tract, bone, bone marrow, reproductivetrack, eye, cartilage, stomach, skin, spinal cord, bladder, salivarygland, and other types of tissues were obtained with a microtome,allowed to desiccate in a freezer, and exposed to phosphoimager platesfor about ten days.

These plates were developed, and the signal (densitometry) from eachorgan was normalized to the signal found in the heart blood of eachanimal. A signal in tissue darker than the signal expected from blood inthat tissue indicates peptide accumulation in a region, tissue,structure or cell. For instance, the cartilage is avascular and containsminute amounts of blood. A ratio of at least 170% signal in thecartilage versus heart ventricle was chosen as a reference level forsignificant targeting to cartilage, which also correlated with clearaccumulation in cartilaginous tissues in the images of the slices. FIG.34 identifies the locations of the SEQ ID NO: 511 peptide distributionin joint and other cartilage. FIG. 45 identifies the locations of theSEQ ID NO: 511 peptide distribution in nasal, spinal, tracheal, andother cartilage, including to hyaline cartilage such as articularcartilage and physeal cartilage, as well as fibrocartilage.

Additionally, the peptide can be retained in cartilage for hours aftertreatment. The SEQ ID NO: 511 peptide was radiolabeled as in EXAMPLE 39and 100 nmol of peptide was injected into a mouse with intact kidneys.FIG. 42 illustrates the retention of and the tissue distribution in thecartilage of a peptide of SEQ ID NO: 511, 24 hours after administration.

Example 41 Dosing of Peptide without Kidney Ligation

This example describes a dosing scheme for administering peptides tomice without kidney ligation. The peptide administered had the sequenceof SEQ ID NO: 511 as shown in TABLE 51. The peptide was radiolabeled bymethylating lysines and the N-terminus, so the actual binding agent maycontain methyl or dimethyl lysine(s) and a methylated or dimethylatedamino terminus.

A target dosage of 100 nmol of each peptide carrying 10-25 μCi of ¹⁴Cwas administered to Female Harlan athymic nude mice by a tail veininjection. Each peptide was allowed to freely circulate within theanimal for either 4 hours or 24 hours before the animals were euthanizedand sectioned.

Example 42 Peptide Homing with Intact Kidneys

This example illustrates peptide homing to cartilage in animals withintact kidneys. At the end of the 4 hour or 24 hour dosing periods inEXAMPLE 41, mice were frozen in a hexane/dry ice bath and then frozen ina block of carboxymethylcellulose. Whole animal sagittal slices wereprepared that resulted in thin frozen sections being available forimaging. Thin, frozen sections of animal including imaging of tissuessuch as brain, tumor, liver, kidney, lung, heart, spleen, pancreas,muscle, adipose, gall bladder, upper gastrointestinal track, lowergastrointestinal track, bone, bone marrow, reproductive track, eye,cartilage, stomach, skin, spinal cord, bladder, salivary gland, andother types of tissues were obtained with a microtome, allowed todesiccate in a freezer, and exposed to phosphoimager plates for aboutten days.

These plates were developed. A signal in tissue darker than the signalexpected from blood in that tissue indicates peptide accumulation in aregion, tissue, structure or cell. For instance, the cartilage isavascular and contains minute amounts of blood. High signal in thekidneys indicates presence and accumulation of the peptide in thekidneys. FIG. 34 identifies the locations of the SEQ ID NO: 511 peptidedistribution in joint and other cartilage as well as kidneys.

Example 43 Peptide Homing with Therapeutic Agents

This example describes certain exemplary therapeutic agents that areconjugated to a peptide. A peptide of the disclosure is expressedrecombinantly or chemically synthesized and then is conjugated to anexemplary drug, such as paclitaxel or triamcinolone acetonide orbudesonide using techniques known in the art, such as those described inBioconjugate Techniques by Greg Hermanson (Elsevier Inc., 3^(rd)Edition, 2013). One or more drugs is conjugated per peptide, or anaverage of less than one drug is conjugated per peptide.

Coupling of these drugs to a peptide of any of SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048 targets the drug to the cartilageof the subject. One or more drug-peptide conjugates are administered toa human or animal.

Example 44 Peptide Homing to an Arthritic Joint

This example illustrates peptide homing to cartilage in humans oranimals with arthritis. A peptide of the present disclosure is expressedrecombinantly or chemically synthesized and is used directly, afterradiolabeling, or after conjugation to a fluorophore or therapeuticcompound. A peptide is selected from any one of the peptides of SEQ IDNO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. The peptide orpeptide conjugate is administered to a human or animal subcutaneously,intravenously, or orally, or is injected directly into a jointintraarticularly. The peptide or peptide conjugate homes to cartilage.

Example 45 Peptide Homing to Cartilage in Non-Human Animals

This example illustrates a peptide or peptide conjugate of thisdisclosure homing to cartilage in non-human animals. Non-human animalsinclude but are not limited to guinea pigs, rabbits, dog, cats, horses,rats, mice, cows, pigs, non-human primates, and other non-human animals.A peptide of the present disclosure is recombinantly expressed orchemically synthesized and are used directly, after radiolabeling, orafter conjugation to a fluorophore or therapeutic compound. The peptideis selected from any one of the peptides of SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. The resulting peptide or peptideconjugate is administered to a non-human animal subcutaneously,intravenously, or orally, or is injected directly into a jointintra-articularly. Biodistribution is assessed by LC/MS,autoradiography, positron emission tomography (PET), or fluorescenceimaging. A peptide or peptide conjugate is homed to cartilage innon-human animals.

Example 46 Whole Body Fluorescence and Isolated Limb Fluorescence ofHoming Peptides

This example illustrates whole body fluorescence and isolated limbfluorescence of peptide homers of this disclosure. Any peptide of thepresent disclosure is chemically conjugated to one molecule of a nearinfrared fluorophore, at the N-terminus of the peptide via an active NHSester on the dye. A dose of 10 nmol of each peptide conjugated to afluorophore is administered to Female Harlan athymic nude mice, weighing20-25 g, and is administered via tail vein injection. Each experiment isdone at least in duplicate (n=2 mice per group). The peptide fluorophoreconjugate is allowed to freely circulate for the described time periodbefore the mice were euthanized at various time points. Mice areevaluated for peptide distribution of the peptide fluorescence in wholebody imaging and in isolated hind limb imaging.

For Whole body fluorescence (WBF), at the end of the dosing period, miceare frozen in a hexane/dry ice bath and then embedded in a frozen blockof carboxymethylcellulose. Whole animal sagittal slices are preparedthat resulted in thin frozen sections for imaging. Thin frozen sectionsare obtained using a microtome and allowed visualization of tissues.Sections are allowed to dessicate in a freezer prior to imaging. WBF isperformed on fluorescent sections, which are scanned on a Li-Cor Odysseyscanner at a setting of 169 μm resolution, medium quality, 700 channel,L-2.0 intensity.

For isolated hind limb fluorescence studies, mice are euthanized by CO₂asphyxiation at the end of the dosing period. The right hind limb isremoved at the hip joint and imaged on a Sepctrum IVIS imager (ex/em:675 nm. 720 nm) with a 1 second exposure length and a focal height of0.5 cm. Limbs are imaged with skin removed and with muscle removed.

Example 47 Whole Body Autoradiography of Homing Peptides

This example illustrates whole body autoradiography of peptide homers ofthis disclosure. Peptides are radiolabeled by methylating lysines at theN-terminus as described in EXAMPLE 35. As such, the peptide may containmethyl or dimethyl lysines and a methylated or dimethlyated aminoterminus. A dose of 100 nmol radiolabeled peptide is administered viatail vein injection in Female Harlan athymic nude mice, weighing 20-25g. The experiment is done in at least duplicate (n=2 animals per group).In some animals, kidneys are ligated to prevent renal filtration of theradiolabled peptides and extend plasma half-life. Each radiolabeledpeptide is allowed to freely circulate within the animal for thedescribed time period before the animals were euthanized and sectioned.

Whole body autoradiography (WBA) sagittal sectioning is performed asfollows. At the end of the dosing period, mice are frozen in ahexane/dry ice bath and then embedded in a frozen block ofcarboxymethylcellulose. Whole animal sagittal slices are prepared thatresulted in thin frozen sections for imaging. Thin frozen sections areobtained using a microtome and allowed visualization of tissues such asbrain, tumor, liver, kidney, lung, heart, spleen, pancreas, muscle,adipose, gall bladder, upper gastrointestinal tract, lowergastrointestinal tract, bone, bone marrow, reproductive tract, eye,cartilage, stomach, skin, spinal cord, bladder, salivary gland, andmore. Sections are allowed to dessicate in a freezer prior to imaging.

For the autoradiography imaging, tape mounted thin sections are freezedried and radioactive samples were exposed to phosphoimager plates for 7days. These plates are developed and the signal (densitometry) from eachorgan was normalized to the signal found in the cardiac blood of eachanimal. A signal in tissue darker than the signal expected from blood inthat tissue indicates accumulation in a region, tissue, structure, orcell.

Example 48 Peptide Localization in Chondrocytes

This example illustrates binding of peptides of this disclosure tochondrocytes within cartilage in animals with intact kidneys. In oneembodiment, animals are dosed and are processed as described in EXAMPLE46 and EXAMPLE 47. At the end of the dosing period, animals areeuthanized and cartilage is optionally removed for use in staining andimaging procedures. Whole animal sagittal slices are prepared thatresult in thin frozen sections being available for staining and imaging.One or more of the following cartilage components are identified in thinfrozen sections or live cartilage explants using standard stainingtechniques: collagen fibrils, glycosaminoglycans, or chondrocytes. Apeptide of this disclosure is found to localize to chondrocytes incartilage, localized intracellularly or extracellularly bound or both.Localization is visualized and confirmed by microscopy.

In another embodiment, peptides or peptide-drug conjugates of thisdisclosure are administered in humans and are localized on or inchondrocytes in cartilage.

Example 49 Peptide Localization in Cartilage Extracellular Matrix

This example illustrates localization of peptides of this disclosure incartilage extracellular matrix. In one embodiment, animals are dosed andare processed as described in EXAMPLE 46 and EXAMPLE 47 in animals withintact kidneys. At the end of the dosing period, animals are euthanizedand cartilage is optionally removed for use in staining and imagingprocedures. Whole animal sagittal slices are prepared that result inthin frozen sections being available for staining and imaging. Thinfrozen sections or live cartilage explants are acquired, stained, andvisualized as described in EXAMPLE 48. A peptide of the presentdisclosure is found to localize to the extracellular matrix incartilage. The peptide may be bound to one or more components of theextracellular matrix, such as proteoglycans, glycosaminoglycans,aggrecan, decorin, or collagen. Localization is visualized and confirmedby microscopy.

In another embodiment, peptides or peptide-drug conjugates of thisdisclosure are administered in humans and are localized in cartilageextracellular matrix.

Example 50 Peptide Binding to Cartilage Explants

This example illustrates a peptide or peptide conjugation of thisdisclosure homing, targeting, being directed to, migrating to, beingretained by, accumulating in, or binding to human and animal cartilageexplants in culture. A peptide is selected from any one of the peptidesof SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048.Peptides are recombinantly expressed or chemically synthesized and areused directly, after radiolabeling, or after conjugation to afluorophore or therapeutic compound. A peptide of peptide conjugate ofthis disclosure is incubated with cartilage explants derived from humansor animals. Peptides of peptide conjugate are found to bind to cartilageexplants. The interaction with cartilage is confirmed using variousmethods that include but are not limited to liquid scintillationcounting, confocal microscopy, immunohistochemistry, HPLC, or LC/MS. Thepeptide shows a higher level of signal than a control peptide that isadministered that is not a cartilage binding peptide.

Example 51 Effects of Peptide on Ion Channels

This example describes the interaction between peptides of the presentdisclosure and ion channels. Ion channels can be associated with painand can be activated in disease states such as arthritis. A peptide ofthe disclosure is expressed and administered in a pharmaceuticalcomposition to a patient to treat a joint condition or diseaseassociated with an ion channel and treatable by binding, blocking, orinteracting with the ion channel Ion channels, such as Nav 1. 7, areinhibited by peptides of the present disclosure. A given peptide isexpressed recombinantly or chemically synthesized, wherein the peptideselected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO:1048. Following expression or synthesis, the peptide is used directly orconjugated to a therapeutic compound, such as those described herein. Apeptide of the present disclosure selectively interacts with ionchannels, or is mutated in order to interact with ion channels. Forexample, a peptide of this disclosure is bound to Nav 1. 7 or Nav 1. 7is blocked by a peptide of this disclosure. When the peptide isadministered to a human subject, Nav 1.7 signaling is reduced in thetissues in proximity to the joints, and pain relief is thereby provided.

Example 52 Peptide-Fc Protein Fusions

This example illustrates making and using peptide-Fc protein fusions. Apeptide of SEQ ID NO: 592 was recombinantly expressed with the sequencefor the human IgG1 Fc protein in HEK293 cells to yield a sequence of SEQID NO: 1049

(METDTLLLWVLLLWVPGSTGGSGVPINVRCRGSRDCLDPCRRAGMRFGRCINSRCHCTPGGSGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).

The sequence of any peptide of this disclosure is expressed as a fusionprotein with either murine or human Fc by adding a secretion signalsequence to the N-terminus and an Fc sequence to the C-terminus. Thiscreates a bivalent molecule with improved secretion properties. Thelarger peptide-Fc fusion is expressed in different mammalian or insectcell lines and is useful as a research reagent and a therapeutic.

Fc fusion to a peptide of SEQ ID NO: 592 to yield a sequence of SEQ IDNO: 1049 extends half-life and improves biodistribution of the peptideto cartilage. Any peptide of this disclosure is co-expressed with Fcprotein to yield Fc-fusion peptides with longer half-life and improvedhoming to cartilage. In SEQ ID NO: 1049, the secretion signal sequenceMETDTLLLWVLLLWVPGSTG (SEQ ID NO: 1050) is followed by the peptide of SEQID NO: 592, and is followed by the sequence for Fc protein. Cleaving canbe imprecise, resulting in cleavage at position 20 or position 21 of SEQID NO: 1049.

Example 53 Peptide Conjugate Hydrolysis

This example describes preparation of peptide conjugates having tunablehydrolysis rates. The peptide-drug conjugates described below aresynthesized with the modification that instead of using succinicanhydride, other molecules are used to provide steric hindrance tohydrolysis or an altered local environment at the carbon adjacent to thefinal hydrolyzable ester. In one exemplary conjugate, the peptide-drugconjugate is synthesized with tetramethyl succinic anhydride to generatehindered esters, which causes a decreased rate of hydrolysis. In anotherexemplary conjugate, one methyl group is present at the adjacent carbon.In another exemplary conjugate, two methyl groups are present at theadjacent carbon. In another exemplary conjugate, one ethyl group ispresent at the adjacent carbon. In another exemplary conjugate, twoethyl groups are present at the adjacent carbon. In another exemplaryconjugate, the carbon linker length is increased such as by usingglutaric anhydride instead of succinic anhydride, increasing the localhydrophobicity and lowering the hydrolysis rate. In another exemplaryconjugate, a hydroxyl group is located on the adjacent carbon,increasing the local hydrophilicity and increasing the hydrolysis rate.The rate of hydrolysis in these exemplary conjugates is thereforeadjusted, preventing premature cleavage and ensuring that the majorityof peptide-dexamethasone conjugates accumulate in cartilage prior torelease of the drug by hydrolysis but that the dexamethasone is alsoreleased in the cartilage in a timely manner.

The resulting peptide conjugates are administered to a human or animalsubcutaneously, intravenously, orally, or injected directly into a jointto treat disease.

Example 54 Peptide Conjugates with Stable Linkers

This example describes preparation of peptide conjugates with stablelinkers. A peptide of the disclosure is expressed recombinantly or ischemically synthesized. The peptide is conjugated to a detectable agentor an active agent via a stable linker, such as an amide linkage or acarbamate linkage. The peptide is conjugated to a detectable agent or anactive agent via a stable linker, such as an amide bond using standard1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) ordicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride orphosphorous chloride-based bioconjugation chemistries.

A peptide and drug conjugated via a linker are described with theformula Peptide-A-B-C-Drug, wherein the linker is A-B-C. A can be astable amide link that is formed by reacting with an amine on thepeptide with a linker containing a tetrafluorophenyl (TFP) ester or anNHS ester. A can also be a stable carbamate linker that is formed byreacting with an amine on the peptide imidazole carbamate activeintermediate formed by the reaction of CDI with a hydroxyl on thelinker. A can also be a stable secondary amine linkage that is formed byreductive alkylation of the amine on the peptide with an aldehyde orketone group on the linker. A can also be a stable thioether linkerformed using a maleimide or bromoacetamide in the linker with a thiol inthe peptide, a triazole linker, a stable oxime linker, or anoxacarboline linker. B is (—CH₂—)_(x)—, a short PEG (—CH₂CH₂O—)_(x) (xis 0-20). Alternatively, spacers within the linker is optional and canbe included or not at all. C is an amide bond formed with an amine or acarboxylic acid on the drug, a thioether formed between a maleimide onthe linker and a sulihydroyl on the drug, a secondary or tertiary amine,a carbamate, or other stable bonds. Any linker chemistry described in“Current ADC Linker Chemistry,” Jain et al., Pharm Res, 2015 DOI 10.1007/s11095-015-1657-7 can be used.

The resulting peptide conjugates are administered to a human or animalsubcutaneously, intravenously, orally, or injected directly into a jointto treat disease. The peptide is not specifically cleaved from thedetectable agent or active agent via a targeted mechanism. The peptidecan be degraded by mechanisms such as catabolism, releasing a drug thatis modified or not modified form its native form (Singh, Luisi, and Pak,Pharm Res 32:3541-3571 (2015)). The peptide drug conjugate exerts itspharmacological activity while still intact, or while partially or fullydegraded, metabolized, or catabolized.

Example 55 Peptide Conjugates with Cleavable Linkers

This example describes preparation of peptide conjugates havingcleavable linkers. A peptide of the disclosure is expressedrecombinantly or chemically synthesized. A peptide and drug areconjugated via a linker and is described with the formulaPeptide-A-B-C-Drug, wherein the linker is A-B-C. A is a stable amidelink such as that formed by reacting an amine on the peptide with alinker containing a tetrafluorophenyl (TFP) ester or an NHS ester. A canalso be a stable carbamate linker such as that formed by reacting anamine on the peptide with an imidazole carbamate active intermediateformed by reaction of CDI with a hydroxyl on the linker. A can also be astable secondary amine linkage such as that formed by reductivealkylation of the amine on the peptide with an aldehyde or ketone groupon the linker. A can also be a stable thioether linker formed using amaleimide or bromoacetamide in the linker with a thiol in the peptide, atriazole linker, a stable oxime linker, or a oxacarboline linker. B is(—CH₂—)_(x)— or a short PEG (—CH₂CH₂O—)_(x) (x is 0-20) or other spacersor no spacer. C is an ester bond to the hydroxyl or carboxylic acid onthe drug, or a carbonate, hydrazone, or acylhydrazone, designed forhydrolytic cleavage. The hydrolytic rate of cleavage is varied byvarying the local environment around the ester, including carbon length(—CH₂—)x, steric hindrance (including adjacent side groups such asmethyl, ethyl, cyclic), hydrophilicity or hydrophobicity. Hydrolysisrate is affected by local pH, such as lower pH in certain compartmentsof the body or of the cell such as endosomes and lysosomes or diseasedtissues. C is a pH sensitive group such as a hydrazone or oxime linkage.Alternatively C is a disulfide bond designed to be released byreduction, such as by glutathione. Alternatively C (or A-B-C) is apeptidic linkage design for cleavabe by enzymes. Optionally, aself-immolating group such as pABC is included to cause release of afree unmodified drug upon cleavage (Antibody-Drug Conjugates: Design,Formulation, and Physicochemical Stability, Singh, Luisi, and Pak. PharmRes (2015) 32:3541-3571). The linker is cleaved by enzymes such asesterases, matrix metalloproteinases, cathepsins such as cathepsin B,glucuronidases, a protease, or thrombin. Alternatively, the bonddesigned for cleavage is at A, rather than C, and C could be a stablebond or a cleavable bond. An alternative design is to have stablelinkers (such as amide or carbamate) at A and C and have a cleavablelinker in B, such as a disulfide bond. The rate of reduction ismodulated by local effects such as steric hindrance from methyl or ethylgroups or modulating hydrophobicity/hydrophilicity.

The resulting peptide conjugates are administered to a human or animalsubcutaneously, intravenously, orally, or injected directly into a jointto treat disease.

Example 56 Acetylsalicylic Acid Peptide Conjugate

This example describes the conjugation of acetylsalicylic acid to apeptide using a lactic acid linker. A conjugate is produced from amixture of (R,S)— acetylsalicylic acid, lactic acid, and a peptide:

The acetylsalicylic acid -lactic acid linker conjugate depicted above isthen reacted with a lysine or the N-terminus of a cystine-dense peptideto create an acetylsalicylic acid -lactic acid-peptide conjugate. Thecystine-dense peptide is selected from the peptides of SEQ ID NO:508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048.

Acetylsalicylic acid is currently dosed as an enantiomeric mixture, inwhich enantiomers with a single racemic stereocenter are very difficultto separate. As in the reaction scheme (I), a diastereomer with twochiral centers is created by the addition of a chiral linker such asL-lactic acid. Since diastereomers are easily separated, the activeenantiomer of acetylsalicylic acid conjugated to the lactic acid linkercan be purified prior to conjugation to a cystine-dense peptide. Thechemical synthesis can use any conjugation techniques known in the art,such as described in Bioconjugate Techniques by Greg Hermanson and in“Ketorolac-dextran conjugates: synthesis, in vitro, and in vivoevaluation:” Acta Pharm. 57 (2007) 441-450, Vyas, Trivedi, andChaturvedi. The conjugate can display anti-inflammatory activity, orfree acetylsalicylic acid is released from the conjugate to provideanti-inflammatory activity. The free acetylsalicylic acid can resultfrom hydrolysis that occurs after administration, such as hydrolysis atthe ester bond. By dosing the conjugate containing the cartilage homingpeptide, a higher AUC of acetylsalicylic acid delivery to the joint maybe achieved than would be achieved by systemic dosing of acetylsalicylicacid alone.

Such peptide-drug conjugates can be made using either a cleavable orstable linker as described herein (e. g., EXAMPLES 54 and 55).

Example 57 Acetylsalicylic Acid Peptide Conjugate

This example describes the conjugation of acetylsalicylic acid to apeptide using a PEG linker. A conjugate is produced usingacetylsalicylic acid and a PEG linker, which forms an ester bond thatcan hydrolyze as described in “In vitro and in vivo study ofpoly(ethylene glycol) conjugated ibuprofen to extend the duration ofaction,” Scientia Pharmaceutica, 2011, 79:359-373, Nayak and Jain.Fischer esterification is used to conjugate ibuprofen with a short PEG,e.g., with triethylene glycol, to yield ibuprofen-ester-PEG-OH.

Following preparation of the PEG-ibuprofen conjugate as shown above, thehydroxyl moiety of PEG is activated with N,N′-disuccinimidyl carbonate(DSC) to form ibuprofen-ester-PEG-succinimidyl carbonate, which is thenreacted with a lysine or the N-terminus of a cystine-dense peptide toform an ibuprofen-ester-PEG-peptide conjugate. The cystine-dense peptideis selected from any one of the peptides of sequence SEQ ID NO: 508—SEQID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. The conjugate can displayanti-inflammatory activity, or free ibuprofen is released from theconjugate to provide anti-inflammatory activity. The free ibuprofen canresult from hydrolysis that occurs after administration, such ashydrolysis at the ester bond.

Ibuprofen-peptide conjugates are administered to a subject in needthereof. The subject can be a human or a non-human animal.

Such peptide-drug conjugates can be made using either a cleavable orstable linker as described herein (e.g., EXAMPLES 54 and 55).

Example 58 Dexamethasone Peptide Conjugate

This example describes different methods of conjugating dexamethasonewith a peptide of this disclosure. A peptide of SEQ ID NO: 592 wasrecombinantly expressed. Dexamethasone was readily conjugated to apeptide of this disclosure using a dicarboxylic acid linker. Thepeptide-dexamethasone conjugate was made by first convertingdexamethasone to a hemisuccinate by reacting it with succinic anhydride.The hemisuccinate was then converted to a succinate carboxylic acidcontaining an active ester, using dicyclohexyl carbodiimide (DCC) or1-ethyl-3-(3-dimethylamninopropyl)carbodiimide (EDC) in the presence ofN-hydroxy succinimide (NHS). This active ester was then reacted with alysine or the N-terminus of a cystine-dense peptide to create adexamethasone-carboxylic acid-peptide conjugate. Methods such as thosedescribed in “Functionalized derivatives of hyaluronic acidoligosaccharides: drug carriers and novel biomaterials” BioconjugateChemistry 1994, 5, 339-347, Pouyani and Prestwich, and BioconjugateTechniques by Greg Hermanson can be used (Elsevier Inc., 3^(rd) Edition,2013).

Peptide-dexamethasone conjugates were prepared by coupling dexamethasoneto the peptides of this disclosure using standard coupling-reagentchemistry. For example, dexamethasone conjugates were made by reactingdexamethasone hemigluterate with 1.05 molar equivalents of1,1′-carbonyldiimidazole in anhydrous DMSO in an inert atmosphere. After30 minutes, excess dexamethasone in anhydrous DMSO was added along withtwo molar equivalents of anhydrous trimethylamine. TheN-hydroxysuccinimide ester of the peptide-dexamethasone conjugate wasgenerated to form a shelf-stable intermediate for later reaction with anamine-containing carrier. The N-terminal dexamethasone-peptide conjugate(SEQ ID NO: 592B) was verified by electrospray mass spectrometry (ES-MS)within a 10 ppm error.

A peptide of any of the sequences of this disclosure including SEQ IDNO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048, are conjugatedto dexamethasone using the methods described above.

Such peptide-drug conjugates can be made using either a cleavable orstable linker as described herein (e.g., EXAMPLES 54 and 55).

Example 59 Beclomethasone Monopropionate Peptide Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 511 or SEQID NO: 592 of this disclosure to beclomethasone monopropionate.Beclomethasone monopropionate is readily conjugated to any peptidedisclosed herein via a dicarboxylic acid linker. The dicarboxylic acidlinker is a linear dicarboxylic acid, such as succinic acid, or arelated cyclic anhydride, such as succinic anhydride. Reactions withanhydrides can proceed under simple conditions. For example, thereaction of beclomethasone monopropionate with five molar equivalents ofglutaric anhydride is carried out in anhydrous pyridine at roomtemperature. Reactions with dicarboxylic acids can occur using standardcarbodiimide coupling methods. For example, beclomethasonemonopropionate is reacted with one molar equivalent dimethylsuccinicacid, one molar equivalent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(or another carbodiimide), and 0.2 molar equivalents of 40-dimethylaminopyridine.

The same methods as described in EXAMPLE 53 are used to adjust the rateof hydrolysis of peptide-beclomethasone monopropionate conjugates,preventing premature cleavage and ensuring that the beclomethasonemonopropionate of peptide-beclomethasone monopropionate conjugatesaccumulate in cartilage.

Peptide-beclomethasone monopropionate conjugates are prepared bycoupling beclomethasone monopropionate to the peptides of thisdisclosure using standard coupling-reagent chemistry. Thepeptide-beclomethasone monopropionate conjugate was made by firstconverting beclomethasone monopropionate to a hemisuccinate by reactingit with succinic anhydride. The hemisuccinate was then converted to asuccinate carboxylic acid containing an active ester, using dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylamninopropyl)carbodiimide(EDC) in the presence of N-hydroxy succinimide (NHS). This active esterwas then reacted with a lysine or the N-terminus of a peptide to createa beclomethasone monopropionate -carboxylic acid-peptide conjugate.Methods such as those described in “Functionalized derivatives ofhyaluronic acid oligosaccharides: drug carriers and novel biomaterials”Bioconjugate Chemistry 1994, 5, 339-347, Pouyani and Prestwich, andBioconjugate Techniques by Greg Hermanson can be used (Elsevier Inc.,3^(rd) Edition, 2013).

Peptide-beclomethasone monopropionate conjugates were prepared bycoupling beclomethasone monopropionate to the peptides of thisdisclosure using standard coupling-reagent chemistry. For example,beclomethasone monopropionate conjugates were made by reactingbeclomethasone monopropionate hemigluterate with 1.05 molar equivalentsof 1,1′-carbonyldiimidazole in anhydrous DMSO in an inert atmosphere.After 30 minutes, excess beclomethasone monopropionate in anhydrous DMSOwas added along with two molar equivalents of anhydrous trimethylamine.The N-hydroxysuccinimide ester of the peptide-beclomethasonemonopropionate conjugate was generated to form a shelf-stableintermediate for later reaction with an amine-containing carrier.

Beclomethasone monopropionate is also readily conjugated to any peptidedisclosed herein via a dicarboxylic acid linker. The dicarboxylic acidlinker is a linear dicarboxylic acid, such as succinic acid, or arelated cyclic anhydride, such as succinic anhydride. Reactions withanhydrides can proceed under simple conditions. For example, thereaction of beclomethasone monopropionate with five molar equivalents ofglutaric anhydride is carried out in anhydrous pyridine at roomtemperature. Reactions with dicarboxylic acids can occur using standardcarbodiimide coupling methods. For example, beclomethasonemonopropionate is reacted with one molar equivalent dimethylsuccinicacid, one molar equivalent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(or another carbodiimide), and 0.2 molar equivalents of 40-dimethylaminopyridine. The peptide-beclomethasone monopropionate conjugates areadministered to a subject in need thereof and home, target, are directedto, are retained by, accumulate in, migrate to, and/or bind to cartilageand/or kidneys. The subject is a human or animal and has inflammation inthe cartilage or kidney tissues. Upon administration of thepeptide-beclomethasone monopropionate conjugates, the cartilage and/orkidney inflammation is alleviated.

The peptide can also be a peptide of SEQ ID NO: 517. The peptide can beany peptide with the sequence selected SEQ ID NO: 508—SEQ ID NO: 758 orSEQ ID NO: 798—SEQ ID NO: 1048.

Such peptide-drug conjugates are made using either a cleavable or stablelinker as described herein (e.g., EXAMPLES 54 and 55).

Example 60 Desciclesonide Peptide Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 683 or SEQID NO: 671 of this disclosure to desciclesonide. Ciclesonide is aprodrug that is metabolized in vivo to the active metabolitedesciclesonide. By conjugating desciclesonide to a peptide via an esterlinker, upon hydrolysis the released drug would be desciclesonide, justas after systemic administration of ciclesonide the active metabolitedesciclesonide is present and active. Desciclesonide is readilyconjugated to any peptide disclosed herein via a dicarboxylic acidlinker. The dicarboxylic acid linker is a linear dicarboxylic acid, suchas succinic acid, or a related cyclic anhydride, such as succinicanhydride. Reactions with anhydrides can proceed under simpleconditions. For example, the reaction of desciclesonide with five molarequivalents of glutaric anhydride is carried out in anhydrous pyridineat room temperature. Reactions with dicarboxylic acids can occur usingstandard carbodiimide coupling methods. For example, desciclesonide isreacted with one molar equivalent dimethylsuccinic acid, one molarequivalent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (or anothercarbodiimide), and 0.2 molar equivalents of 40-dimethylamino pyridine.

The same methods as described in EXAMPLE 53 are used to adjust the rateof hydrolysis of peptide- desciclesonide conjugates, preventingpremature cleavage and ensuring that the desciclesonide of peptide-desciclesonide conjugates accumulate in cartilage.

Desciclesonide is also readily conjugated to any peptide disclosedherein via a dicarboxylic acid linker. The dicarboxylic acid linker is alinear dicarboxylic acid, such as succinic acid, or a related cyclicanhydride, such as succinic anhydride. Reactions with anhydrides canproceed under simple conditions. For example, the reaction ofdesciclesonide with five molar equivalents of glutaric anhydride iscarried out in anhydrous pyridine at room temperature. Reactions withdicarboxylic acids can occur using standard carbodiimide couplingmethods. For example, desciclesonide is reacted with one molarequivalent dimethylsuccinic acid, one molar equivalent1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (or another carbodiimide),and 0.2 molar equivalents of 40-dimethylamino pyridine. The peptide-desciclesonide conjugates are administered to a subject in need thereofand home, target, are directed to, are retained by, accumulate in,migrate to, and/or bind to cartilage and/or kidneys. The subject is ahuman or animal and has inflammation in the cartilage or kidney tissues.Upon administration of the peptide-desciclesonide conjugates, thecartilage and/or kidney inflammation is alleviated.

The peptide-desciclesonide conjugates are administered to a subject inneed thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to cartilage and/or kidneys. Thesubject is a human or animal and has inflammation in the cartilage orkidney tissues. Upon administration of the peptide-desciclesonideconjugates, the cartilage and/or kidney inflammation is alleviated.

The peptide can also be a peptide of SEQ ID NO: 680. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048.

Such peptide-drug conjugates are made using either a cleavable or stablelinker as described herein (e.g., EXAMPLES 54 and 55).

Example 61 Desciclesonide Peptide Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 683 or SEQID NO: 671 of this disclosure to desciclesonide. Ciclesonide is aprodrug that is metabolized in vivo to the active metabolitedesciclesonide. By conjugating desciclesonide to a peptide via an esterlinker, upon hydrolysis the released drug would be desciclesonide, justas after systemic administration of ciclesonide the active metabolitedesciclesonide is present and active. Desciclesonide is readilyconjugated to any peptide disclosed herein via a stable linker.

The peptide-desciclesonide conjugates are administered to a subject inneed thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to cartilage and/or kidneys. Thesubject is a human or animal and has inflammation in the cartilage orkidney tissues. Upon administration of the peptide-desciclesonideconjugates, the cartilage and/or kidney inflammation is alleviated.

The peptide can also be a peptide of SEQ ID NO: 680. The peptide can beany peptide with the sequence selected SEQ ID NO: 508—SEQ ID NO: 758 orSEQ ID NO: 798—SEQ ID NO: 1048.

Such peptide-drug conjugates are made using either a cleavable or stablelinker as described herein (e.g., EXAMPLES 54 and 55).

Example 62 Peptide-Ustekinumab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 590 thisdisclosure to ustekinumab. Ustekinimab is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013). Alternatively thepeptide-active agent of this Example can be expressed as a fusionprotein. From one to eight peptides are linked to ustekinumab.

The peptide-ustekinumab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has psoriatic arthritis. Upon administration of thepeptide-ustekinumab conjugates, the psoriatic arthritis condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 520. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048.

Example 63 Peptide-Xeljanz Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 671 thisdisclosure to xeljanz. Xeljanz is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked toxeljanz.

The peptide-xeljanz conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has rheumatoid arthritis. Upon administration and homing ofpeptide-xeljanz conjugates, the rheumatoid arthritis condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 669. The peptide can beany peptide with 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 64 Peptide-IL-17 Inhibitor Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to an IL-17 inhibitor. An IL-17 inhibitor is readilyconjugated to any peptide disclosed herein via standard chemistries suchas those described in, but not limited to, Bioconjugate Techniques byGreg Hermanson (Elsevier Inc., 3^(rd) edition, 2013).

The peptide-IL-17 inhibitor conjugates are administered to a subject inneed thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to cartilage. The subject is ahuman or animal and has ankylosing spondylitis. Upon administration andhoming of peptide-IL-17 inhibitor conjugates, the ankylosing spondylitiscondition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 595. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 65 Peptide-Iguratimod Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 683 thisdisclosure to iguratimod. Iguratimod is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013).

The peptide- iguratimod conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to kidneys. The subject is a human or animaland has rheumatoid arthritis. Upon administration and homing ofpeptide-iguratimod conjugates, the rheumatoid arthritis condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 510. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as

Example 66 Peptide Mycophenolic Acid Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 511 thisdisclosure to mycophenolic acid. Mycophenolic acid is readily conjugatedto any peptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013).

The peptide-mycophenolic acid conjugates are administered to a subjectin need thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to kidneys. The subject is ahuman or animal and has organ transplantation, infection, cancer, orother kidney disorders. Upon administration and homing ofpeptide-mycophenolic acid conjugates, the organ transplantation,infection, cancer, other kidney disorders condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 591. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 67 Peptide-Tacrolimus Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to tacrolimus. Tacrolimus is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013).

The peptide-tacrolimusconjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to kidneys. The subject is a human or animaland has organ transplantation, any other kidney disease. Uponadministration and homing of peptide-tacrolimus conjugates, the organtransplantation, any other kidney disease condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 595. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as

Example 68 Peptide-Secukinumab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 590 thisdisclosure to secukinumab. Secukinumab is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013). From one to eightpeptides are linked to secukinumab. Alternatively the peptide-activeagent of this Example can be expressed as a fusion protein.

The peptide-secukinumab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has ankylosing spondylitis. Upon administration and homing ofpeptide-secukinumab acid conjugates, the ankylosing spondylitiscondition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 508. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 69 Peptide-Sirukumab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 683 thisdisclosure to sirukumab. Sirukumab is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked tosirukumab. Alternatively the peptide-active agent of this Example can beexpressed as a fusion protein.

The peptide- sirukumab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to kidneys. The subject is a human or animaland has rheumatoid arthritis, immune diseases of the kidneys. Uponadministration and homing of peptide-sirukumab conjugates, therheumatoid arthritis, immune diseases of the kidneys condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 520. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 70 Peptide-Anifrolumab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 511 thisdisclosure to anifrolumab. Anifrolumab is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013). From one to eightpeptides are linked to anifrolumab. Alternatively the peptide-activeagent of this Example can be expressed as a fusion protein.

The peptide-anifrolumab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to kidneys. The subject is a human or animaland has lupus nephritis. Upon administration and homing ofpeptide-anifrolumab conjugates, the lupus nephritis condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 669. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 71 Peptide-Denosumab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to denosumab. Denosumab is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked todenosumab. Alternatively the peptide-active agent of this Example can beexpressed as a fusion protein.

The peptide-denosumab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has osteoporosis. Upon administration and homing ofpeptide-denosumab conjugates, the osteoporosis condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 509. The peptide can beany peptide with the sequence selected SEQ ID NO: 508—SEQ ID NO: 758 orSEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates can be madeusing either a cleavable or stable linker as described herein (e.g.,EXAMPLES 54 and 55).

Example 72 Peptide-Rituximab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to rituximab. Rituximab is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked torituximab. Alternatively the peptide-active agent of this Example can beexpressed as a fusion protein.

The peptide-rituximab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage or kidneys. The subject is ahuman or animal and has rheumatoid arthritis, kidney transplant. Uponadministration and homing of peptide-rituximab conjugates, therheumatoid arthritis, kidney transplant condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 510. The peptide can beany peptide with the sequence selected SEQ ID NO: 508—SEQ ID NO: 758 orSEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates can be madeusing either a cleavable or stable linker as described herein (e.g.,EXAMPLES 54 and 55).

Example 73 Peptide-Omalizumab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to omalizumab. Omalizumab is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013). From one to eightpeptides are linked to omalizumab. Alternatively the peptide-activeagent of this Example can be expressed as a fusion protein.

The peptide-omalizumab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to kidneys. The subject is a human or animaland has kidney inflammation. Upon administration and homing ofpeptide-omalizumab conjugates, the kidney inflammation condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 591. The peptide can beany peptide with the sequence selected SEQ ID NO: 508—SEQ ID NO: 758 orSEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates can be madeusing either a cleavable or stable linker as described herein (e.g.,EXAMPLES 54 and 55).

Example 74 Peptide-Abatacept Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 683 thisdisclosure to abatacept. Abatacept is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked toabatacept. Alternatively the peptide-active agent of this Example can beexpressed as a fusion protein.

The peptide-abatacept conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to kidneys. The subject is a human or animaland has rheumatoid arthritis, lupus nephritis, organ transplant, focalsegmental glomerulosclerosis. Upon administration and homing ofpeptide-abatacept conjugates, the rheumatoid arthritis, lupus nephritis,organ transplant, focal segmental glomerulosclerosis condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 595. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 75 Peptide-Oxycodone Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 671 thisdisclosure to oxycodone. Oxycodone is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013).

The peptide-oxycodone conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has cartilage or kidney-related pain. Upon administration andhoming of peptide-oxycodone conjugates, the cartilage-related paincondition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 508. The peptide can beany peptide with the sequence selected SEQ ID NO: 508—SEQ ID NO: 758 orSEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates can be madeusing either a cleavable or stable linker as described herein (e.g.,EXAMPLES 54 and 55).

Example 76 Peptide Capsaicin Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to capsaicin. Capsaicin is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013).

The peptide-capsaicin conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has cartilage-related pain. Upon administration and homing ofpeptide-capsaicin conjugates, the cartilage or kidney-related paincondition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 520. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 77 Peptide-GSK2193874 Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 590 thisdisclosure to GSK2193874. GSK2193874 is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013).

The peptide-GSK2193874 conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to GSK2193874. The subject is a human oranimal and has cartilage-related pain. Upon administration and homing ofpeptide-GSK2193874 conjugates, the cartilage-related pain condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 669. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 78 Peptide BIIB023 Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 683 thisdisclosure to BIIB023. BIIB023 is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked toBIIB023. Alternatively the peptide-active agent of this Example can beexpressed as a fusion protein.

The peptide-BIIB023 conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has lupus nephritis or rheumatoid arthritis. Uponadministration and homing of peptide-BIIB023 conjugates, the lupusnephritis or rheumatoid arthritis condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 509. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 79 Peptide-Anakinra Conjugates

This example describes conjugation or fusion of a peptide of SEQ ID NO:671 or SEQ ID NO: 1034—SEQ ID NO: 1048 of this disclosure to anakinra Alinker is optionally used to conjugate the peptide to anakinra Anakinrais readily conjugated to any peptide disclosed herein via standardchemistries such as those described in, but not limited to, BioconjugateTechniques by Greg Hermanson (Elsevier Inc., 3^(rd) edition, 2013). Fromone to eight peptides are linked to anakinra Alternatively thepeptide-active agent of this Example can be expressed as a fusionprotein.

The peptide-anakinra conjugates or fusions are administered to a subjectin need thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to cartilage. The subject is ahuman or animal and has lupus nephritis or rheumatoid arthritis. Uponadministration and homing of peptide-anakinra conjugates or fusions, thelupus nephritis or rheumatoid arthritis condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 510. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 80 Peptide-IGF-1 Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to IGF-1. IGF-1 is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc.^(, 3rd) edition, 2013). From one to eight peptides are linked toIGF-1. Alternatively the peptide-active agent (where the active agent isthe biologic of this Example) can be expressed as a fusion protein.

The peptide-IGF-1 conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has renal cancer or arthritis. Upon administration and homingof peptide-IGF-1 conjugates, the renal cancer or arthritis condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 591. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 81 Peptide-Romosozumab Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 590 thisdisclosure to Romosozumab. Romosozumab is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013). From one to eightpeptides are linked to romosozumab. Alternatively the peptide-activeagent of this Example can be expressed as a fusion protein.

The peptide-romosozumab conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has osteoporosis. Upon administration and homing ofpeptide-romosozumab conjugates, the osteoporosis condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 595. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as

Example 82 Peptide-ZVAD-fmk Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 671 thisdisclosure to ZVAD-fmk. ZVAD-fmk is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked toZVAD-fmk. The peptide-ZVAD-fmk conjugates are administered to a subjectin need thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to cartilage. The subject is ahuman or animal and has cartilage grafting, arthritis, surgicalintervention, surgery for cartilage repair. Upon administration andhoming of peptide- ZVAD-fmk conjugates, the cartilage grafting,arthritis, surgical intervention, surgery for cartilage repair conditionis alleviated.

The peptide can also be a peptide of SEQ ID NO: 508. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 83 Peptide-S-methylisothiourea Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to S-methylisothiourea. S-methylisothiourea is readilyconjugated to any peptide disclosed herein via standard chemistries suchas those described in, but not limited to, Bioconjugate Techniques byGreg Hermanson (Elsevier Inc., 3^(rd) edition, 2013).

The peptide-S-methylisothiourea conjugates are administered to a subjectin need thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to cartilage. The subject is ahuman or animal and has arthritis surgery, kidney iron overload, renalischemia reperfusion injury, or acute kidney injury. Upon administrationand homing of peptide-S-methylisothiourea conjugates, the arthritissurgery, kidney iron overload, renal ischemia reperfusion injury, oracute kidney injury condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 517. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as

EXAMPLE 83 Peptide-P188 Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 590 thisdisclosure to P188. P188 is readily conjugated to any peptide disclosedherein via standard chemistries such as those described in, but notlimited to, Bioconjugate Techniques by Greg Hermanson (Elsevier Inc.,3^(rd) edition, 2013).

The peptide-P188 conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has arthritis surgery. Upon administration and homing ofpeptide-P188 conjugates, the arthritis surgery condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 669. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 84 Peptide-Alendronate Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 671 thisdisclosure to alendronate. Alendronate is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3 ^(rd) edition, 2013).

The peptide-alendronate conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has bone erosion. Upon administration and homing ofpeptide-alendronate conjugates, the bone erosion condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 506. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 85 Peptide-MIP-3α Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to MIP-3α. MIP-3α is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked toMIP-3α. Alternatively the peptide-active agent of this Example can beexpressed as a fusion protein

The peptide-MIP-3α conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has Joint injury, repair and regeneration of cartilage andbone. Upon administration and homing of peptide-MIP-3α conjugates, theJoint injury, repair and regeneration of cartilage and bone condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 510. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 86 Peptide-BMP-2 Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 590 thisdisclosure to BMP-2. BMP-2 is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013). From one to eight peptides are linked toBMP-2. Alternatively the peptide-active agent of this Example can beexpressed as a fusion protein.

The peptide-BMP-2conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has Joint repair. Upon administration and homing ofpeptide-BMP-2conjugates, the Joint repair condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 591. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 87 Peptide-Icariin Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 671 thisdisclosure to icariin. Icariin is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013).

The peptide-icariin conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has Joint repair. Upon administration and homing ofpeptide-icariin conjugates, the Joint repair condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 592. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or a stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 88 Peptide-Captopril Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 511 thisdisclosure to captopril. Captopril is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013).

The peptide-captopril conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to kidneys. The subject is a human or animaland has diabetic nephropathy. Upon administration and homing ofpeptide-captopril conjugates, the diabetic nephropathy condition isalleviated.

The peptide can also be a peptide of SEQ ID NO: 508. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 89 Peptide-Tofacitinib Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to tofacitinib. Tofacitinib is readily conjugated to anypeptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013). From one to eightpeptides are linked to tofacitinib.

The peptide-tofacitinib conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage. The subject is a human oranimal and has rheumatoid arthritis and kidney transplant, ankylosesspondylitis. Upon administration and homing of peptide-tofacitinibconjugates, the rheumatoid arthritis and kidney transplant, ankylosesspondylitis condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 520. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as described herein(e.g., EXAMPLES 54 and 55).

Example 90 Peptide-Dimethyl Fumarate Conjugates

This example describes conjugation of a peptide of SEQ ID NO: 592 thisdisclosure to dimethyl fumarate. Dimethyl fumarate is readily conjugatedto any peptide disclosed herein via standard chemistries such as thosedescribed in, but not limited to, Bioconjugate Techniques by GregHermanson (Elsevier Inc., 3^(rd) edition, 2013). Alternatively,peptide-dimethyl fumarate conjugates can be synthesized by Michaeladdition of a thiol (on the peptide of linker) to dimethyl fumarate asdescribed by Schmidt et al. (Bioorg Med Chem. 2007 Jan. 1; 15(1):333-42.Epub 2006 Sep. 29.).

The peptide-dimethyl fumarate conjugates are administered to a subjectin need thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to kidneys. The subject is ahuman or animal and has Kidney fibrosis, psoriatic arthritis, rheumatoidarthritis. Upon administration and homing of peptide-dimethyl fumarateconjugates, the Kidney fibrosis, psoriatic arthritis, rheumatoidarthritis condition is alleviated.

The peptide can also be a peptide of SEQ ID NO: 671. The peptide can beany peptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates canbe made using either a cleavable or stable linker as

Example 91 Intra-Articular Administration of Peptides and PeptideConjugates

This example illustrates intra-articular administration of peptides orpeptide conjugates of this disclosure. A peptide of this disclosure isexpressed recombinantly or chemically synthesized. In some cases, thepeptide is subsequently conjugated to a detectable agent or an activeagent. The peptide or peptide conjugate is administered to a subject inneed thereof via intra-articular administration. The cartilage ispenetrated by the peptide or peptide conjugate due to the small size ofthe peptide or peptide conjugate, and due to binding of cartilagecomponents by the peptide or peptide conjugate. The peptide or peptideconjugate is bound to cartilage and the residence time in the cartilageis longer due to this binding. Optionally, the injected material isaggregated, is crystallized, or complexes are formed, further extendingthe depot effect and contributing to longer residence time.

The peptide can be a peptide of SEQ ID NO: 592. The peptide can also bea peptide of SEQ ID NO: 508. The peptide can be any peptide with thesequence selected SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ IDNO: 1048. Such peptide-drug conjugates can be made using either acleavable or stable linker as described herein (e.g., EXAMPLES 54 and55).

Example 92 Treatment of Osteoarthritis

This example describes a method for treating osteoarthritis usingpeptides of the present disclosure. This method is used as a treatmentfor acute and/or chronic symptoms associated with osteoarthritis. Apeptide of the present disclosure is expressed recombinantly orchemically synthesized and then is used directly or conjugated to ananti-inflammatory compound, such as aspirin, desciclesonide, orsecukinumab. The resulting peptide or peptide-drug conjugate isadministered in a pharmaceutical composition subcutaneously,intravenously, or orally, or is injected directly into a joint of apatient and targeted to cartilage. The formulation can be modifiedphysically or chemically to increase the time of exposure in thecartilage. One or more anti-inflammatory peptide conjugates areadministered to a human or animal.

The peptide can be a peptide of SEQ ID NO: 590. The peptide can also bea peptide of SEQ ID NO: 517. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 93 Treatment of Cartilage Degradation

This example describes a method for treating and/or preventing cartilagedegradation using a peptide of the present disclosure. This method isused as a treatment for acute and/or chronic symptoms associated withcartilage degradation. Progressive degradation or thinning of thecartilage is difficult to treat in part because molecules such as smallmolecule drugs and antibodies typically do not reach the avascularcartilage. A peptide of the present disclosure is used for its homingand/or native activity, or is mutated to generate activity such as MMPprotease inhibition. It is expressed recombinantly or chemicallysynthesized and then is used directly or conjugated to an extracellularmatrix targeting active agent, such as an inhibitor of MMP activity oran anti-apoptosis agent (e.g., osteoprotegrin, romosozumab, P188,ZVAD-fmk, quercetin, dasatinib, dimethyl fumarate, bortezomib,carilzomib, or navitoclax). The resulting peptide or peptide-drugconjugate is administered in a pharmaceutical compositionsubcutaneously, intravenously, or orally, or is injected directly into ajoint of a patient and targeted to extracellular matrix. One or moreextracellular matrix targeting conjugates are administered to a human oranimal.

The peptide can be a peptide of SEQ ID NO: 671. The peptide can also bea peptide of SEQ ID NO: 511. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 94 Treatment of a Cartilage Injury

This example describes a method for treating a cartilage injury using apeptide of the present disclosure. A peptide of the present disclosureis expressed recombinantly or chemically synthesized and then is useddirectly or conjugated to a therapeutic compound, such as thosedescribed herein, including, but not limited to BMP-2, BMP-7, BMP-9,BMP-13, PDGF, PTH, PTHrP, IL-8, MIP-3α. The resulting peptide orpeptide-drug conjugate is administered in a pharmaceutical compositionto a patient and targeted to cartilage. One or more therapeuticcompound-peptide conjugates are administered to a human or animal.

The peptide can be a peptide of SEQ ID NO: 592. The peptide can also bea peptide of SEQ ID NO: 669. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 95 Treatment of Rheumatoid Arthritis

This example describes a method for treating rheumatoid arthritis. Thismethod is used as a treatment for acute and/or chronic symptomsassociated with rheumatoid arthritis. A peptide of the presentdisclosure is expressed recombinantly or chemically synthesized and thenis used directly, or is conjugated to an anti-inflammatory compound,such as adalimumab, certolizumab, golimumab, thalidomide, lenalidomide,pomalidomide, pentocifylline, bupropion. When the peptide is useddirectly, the peptide can, for example, bind or inhibit ion channelssuch as Kv 1. 3. The resulting peptide or peptide-drug conjugate isadministered in a pharmaceutical composition to a patient and istargeted to cartilage. One or more anti-inflammatory compound-peptideconjugates are administered to a human or animal subcutaneously,intravenously, or orally, or is injected directly into a joint

The peptide can be a peptide of SEQ ID NO: 590. The peptide can also bea peptide of SEQ ID NO: 509. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 96 Treatment of Gout

This example describes a method for treating gout using peptides of thepresent disclosure. This method is used as a treatment for acute and/orchronic symptoms associated with gout. A peptide of the presentdisclosure is expressed and administered in a pharmaceutical compositionto a patient as a therapeutic for gout. A peptide of the disclosure isrecombinantly or chemically synthesized and then is used directly orconjugated to pegloticase to treat a cartilage disorder. A peptide ofthe disclosure is recombinantly or chemically synthesized and then isused directly or conjugated to probenecid to treat a kidney disorder.The peptide is administered in a pharmaceutical composition to a patientand the peptide is targeted to the cartilage or kidney affected by gout.One or more peptides are administered to a human or animalsubcutaneously, intravenously, or orally, or is injected directly into ajoint.

The peptide can be a peptide of SEQ ID NO: 671. The peptide can also bea peptide of SEQ ID NO: 508. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 97 Treatment or Management of Pain

This example describes a method for treating or managing pain associatedwith a cartilage injury or disorder. This method is used as a treatmentfor acute and/or chronic symptoms associated with a cartilage injury ordisorder. A peptide of the disclosure is expressed and administered in apharmaceutical composition to a patient as a therapeutic for pain as aresult of injury or other cartilage or joint condition as describedherein. The peptide of the present disclosure inhibits ion channels,such as Nav 1.7. The peptide is expressed recombinantly or chemicallysynthesized, wherein the peptide selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Alternatively, the peptides ofSEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048 aremutated to maintain the cartilage homing function, but to add orincrease ion channel inhibition, such as to Nav 1. 7. Followingexpression or synthesis, the peptide is used directly or conjugated to anarcotic (e.g., oxycodone), a non-narcotic analgesic, a naturalcounter-irritant (capsaicin), or a pain receptor channel inhibitor (suchas the TRPV4 inhibitor GSK2193874). Following administration of thepeptide, the peptide targets to the cartilage affected by pain. One ormore peptides are administered to a human or animal subcutaneously,intravenously, or orally, or is injected directly into a joint.

The peptide can be a peptide of SEQ ID NO: 592. The peptide can also bea peptide of SEQ ID NO: 591. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 98 Treatment or Management of Pain with Peptides Only

This example describes a method for treating or managing pain associatedwith a cartilage injury or disorder. This method is used as a treatmentfor acute and/or chronic symptoms associated with a cartilage injury ordisorder. A peptide of the disclosure is expressed and administered in apharmaceutical composition to a patient as a therapeutic for pain as aresult of injury or other cartilage or joint condition as describedherein. The peptide of the present disclosure inhibits ion channels,such as Nav 1.7. The peptide is expressed recombinantly or chemicallysynthesized, wherein the peptide selected from SEQ ID NO: 508—SEQ ID NO:758 or SEQ ID NO: 798—SEQ ID NO: 1048. Alternatively, the peptides ofSEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048 aremutated to maintain the cartilage homing function, but to add orincrease ion channel inhibition, such as to Nav 1.7. Followingexpression or synthesis, the peptide is used directly. Followingadministration of the peptide, the peptide targets to the cartilageaffected by pain. One or more peptides are administered to a human oranimal subcutaneously, intravenously, or orally, or is injected directlyinto a joint.

The peptide can be a peptide of SEQ ID NO: 592. The peptide can also bea peptide of SEQ ID NO: 591. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048.

Example 99 Treatment of Chondrosarcoma

This example illustrates treatment of chondrosarcoma using peptides ofthe present disclosure. A peptide of the present disclosure isrecombinantly expressed or chemically synthesized and are used directly,after radiolabeling, or after conjugation to a fluorophore ortherapeutic compound, such as dasatinib. The peptide or peptideconjugate is administered in a pharmaceutical composition to a subjectas a therapeutic for chondrosarcoma. One or more peptides or peptideconjugates of the present disclosure are administered to a subject. Asubject can be a human or an animal. The pharmaceutical composition isadministered subcutaneously, intravenously, orally, or injected directlyinto a joint. The peptides or peptide conjugates target cartilageaffected by chondrosarcoma.

The peptide can be a peptide of SEQ ID NO: 590. The peptide can also bea peptide of SEQ ID NO: 592. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 100 Treatment of Chordoma

This example illustrates treatment of chordoma using peptides of thepresent disclosure. A peptide of the present disclosure is recombinantlyexpressed or chemically synthesized and are used directly, afterradiolabeling, or after conjugation to a fluorophore or therapeuticcompound, such as dasatinib. The peptide or peptide conjugate isadministered in a pharmaceutical composition to a subject as atherapeutic for chordoma. One or more peptides or peptide conjugates ofthe present disclosure are administered to a subject. A subject can be ahuman or an animal. The pharmaceutical composition is administeredsubcutaneously, intravenously, orally, or injected directly into ajoint. The peptides or peptide conjugates target cartilage affected bychordoma.

The peptide can be a peptide of SEQ ID NO: 671. The peptide can also bea peptide of SEQ ID NO: 508. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 101 Treatment for Rapid Pain Relief

This example illustrates rapid pain relief in patients treated forrheumatoid arthritis or osteoarthritis with the peptides or peptideconjugates of this disclosure. A peptide of this disclosure is expressedrecombinantly or chemically synthesized, and then the N-terminus of thepeptide is conjugated to an active agent via an NHS ester to produce apeptide-active agent conjugate. In some aspects the active agent such asa kidney therapeutic from TABLE 54, TABLE 55, or TABLE 56. In somecases, the peptide alone is administered to the subject.

The peptide or peptide-active agent conjugate is administered to asubject in need thereof. The subject is a human or non-human animal. Thesubject in need thereof has rheumatoid arthritis or osteoarthritis. Thepeptide or peptide conjugate is delivered via intravenousadministration. Upon administration, the peptide or peptide conjugaterapidly homes to cartilage. Rapid pain relief within five minutes to anhour is experienced by the subject, and pain relieve can last as long asover 3 hours.

The peptide can be a peptide of SEQ ID NO: 592. The peptide can also bea peptide of SEQ ID NO: 517. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 102 Treatment for Lupus Nephritis

This example illustrates treatment of lupus nephritis using peptides orpeptide conjugates of this disclosure. A peptide of the presentdisclosure is recombinantly expressed or chemically synthesized and areused directly, after radiolabeling, or after conjugation to afluorophore or therapeutic compound, such as abatacept or BIIB023.

The peptide or peptide conjugate is administered in a pharmaceuticalcomposition to a subject as a therapeutic for lupus nephritis. Thepeptide is selected from any one of the peptides of SEQ ID NO: 508—SEQID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. One or more peptides orpeptide conjugates of the present disclosure are administered to asubject. A subject can be a human or an animal. The pharmaceuticalcomposition is administered subcutaneously, intravenously, orally, orinjected directly. The peptides or peptide conjugates target kidneyaffected by lupus nephritis.

The peptide can be a peptide of SEQ ID NO: 511. The peptide can also bea peptide of SEQ ID NO: 508. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 103 Treatment for Acute Kidney Injury (AKI)

This example illustrates treatment of acute kidney injury (AKI) usingpeptides or peptide conjugates of this disclosure. A peptide of thepresent disclosure is recombinantly expressed or chemically synthesizedand are used directly, after radiolabeling, or after conjugation to afluorophore or therapeutic compound, such as such as a kidneytherapeutic from TABLE 54, TABLE 55, or TABLE 56.

The peptide or peptide conjugate is administered in a pharmaceuticalcomposition to a subject as a therapeutic for acute kidney injury (AKI).The peptide is selected from any one of the peptides of SEQ ID NO:508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. One or morepeptides or peptide conjugates of the present disclosure areadministered to a subject. A subject can be a human or an animal. Thepharmaceutical composition is administered subcutaneously,intravenously, orally, or injected directly into a joint. The peptidesor peptide conjugates target cartilage affected by acute kidney injury(AKI).

The peptide can be a peptide of SEQ ID NO: 592. The peptide can also bea peptide of SEQ ID NO: 520. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 104 Treatment for Chronic Kidney Disease (CKD)

This example illustrates treatment of chronic kidney disease (CKD) usingpeptides or peptide conjugates of this disclosure. A peptide of thepresent disclosure is recombinantly expressed or chemically synthesizedand are used directly, after radiolabeling, or after conjugation to afluorophore or therapeutic compound, such as a kidney therapeutic fromTABLE 54, TABLE 55, or TABLE 56.

The peptide or peptide conjugate is administered in a pharmaceuticalcomposition to a subject as a therapeutic for chronic kidney disease(CKD). The peptide is selected from any one of the peptides of SEQ IDNO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. One or morepeptides or peptide conjugates of the present disclosure areadministered to a subject. A subject can be a human or an animal. Thepharmaceutical composition is administered subcutaneously,intravenously, orally, or injected directly into a joint. The peptidesor peptide conjugates target cartilage affected by chronic kidneydisease (CKD).

The peptide can be a peptide of SEQ ID NO: 683. The peptide can be anypeptide with the sequence selected from SEQ ID NO: 508—SEQ ID NO: 758 orSEQ ID NO: 798—SEQ ID NO: 1048. Such peptide-drug conjugates can be madeusing either a cleavable or stable linker as described herein (e.g.,EXAMPLES 54 and 55).

Example 105 Treatment for Hypertensive Kidney Damage

This example illustrates treatment of hypertensive kidney damage usingpeptides or peptide conjugates of this disclosure. A peptide of thepresent disclosure is recombinantly expressed or chemically synthesizedand are used directly, after radiolabeling, or after conjugation to afluorophore or therapeutic compound, such as such as a kidneytherapeutic from TABLE 54, TABLE 55, or TABLE 56.

The peptide or peptide conjugate is administered in a pharmaceuticalcomposition to a subject as a therapeutic for hypertensive kidneydamage. The peptide is selected from any one of the peptides of SEQ IDNO: 508—SEQ ID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. One or morepeptides or peptide conjugates of the present disclosure areadministered to a subject. A subject can be a human or an animal. Thepharmaceutical composition is administered subcutaneously,intravenously, orally, or injected directly into a joint. The peptidesor peptide conjugates target cartilage affected by hypertensive kidneydamage.

The peptide can be a peptide of SEQ ID NO: 511. The peptide can also bea peptide of SEQ ID NO: 669. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 106 Treatment for Diabetic Nephropathy

This example illustrates treatment of diabetic nephropathy usingpeptides or peptide conjugates of this disclosure. A peptide of thepresent disclosure is recombinantly expressed or chemically synthesizedand are used directly, after radiolabeling, or after conjugation to afluorophore or therapeutic compound, such as such as a kidneytherapeutic from TABLE 54, TABLE 55, or TABLE 56.

The peptide or peptide conjugate is administered in a pharmaceuticalcomposition to a subject as a therapeutic for diabetic nephropathy. Thepeptide is selected from any one of the peptides of SEQ ID NO: 508—SEQID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. One or more peptides orpeptide conjugates of the present disclosure are administered to asubject. A subject can be a human or an animal. The pharmaceuticalcomposition is administered subcutaneously, intravenously, orally, orinjected directly into a joint. The peptides or peptide conjugatestarget cartilage affected by diabetic nephropathy.

The peptide can be a peptide of SEQ ID NO: 592. The peptide can also bea peptide of SEQ ID NO: 506. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 107 Treatment for Renal Fibrosis

This example illustrates treatment of renal fibrosis using peptides orpeptide conjugates of this disclosure. A peptide of the presentdisclosure is recombinantly expressed or chemically synthesized and areused directly, after radiolabeling, or after conjugation to afluorophore or therapeutic compound, such as such as a kidneytherapeutic from TABLE 54, TABLE 55, or TABLE 56.

The peptide or peptide conjugate is administered in a pharmaceuticalcomposition to a subject as a therapeutic for renal fibrosis. Thepeptide is selected from any one of the peptides of SEQ ID NO: 508—SEQID NO: 758 or SEQ ID NO: 798—SEQ ID NO: 1048. One or more peptides orpeptide conjugates of the present disclosure are administered to asubject. A subject can be a human or an animal. The pharmaceuticalcomposition is administered subcutaneously, intravenously, orally, orinjected directly into a joint. The peptides or peptide conjugatestarget cartilage affected by renal fibrosis.

The peptide can be a peptide of SEQ ID NO: 683. The peptide can also bea peptide of SEQ ID NO: 510. The peptide can be any peptide with thesequence selected from SEQ ID NO: 508—SEQ ID NO: 758 or SEQ ID NO:798—SEQ ID NO: 1048. Such peptide-drug conjugates can be made usingeither a cleavable or stable linker as described herein (e.g., EXAMPLES54 and 55).

Example 108 Peptide Variants Based on Multiple Sequence Alignment

This example illustrates using multiple sequence alignment to designpeptide variants with increased stability and decreased immunogenicity.An alignment was generated using R language and an “msa” softwarepackage, which codes for R language specific for multiple alignments(Bodenhofer, U et al. Bioinformatics, 31 (24): 3997-3999 (2015)). FIG.44 illustrates a multiple sequence alignment of SEQ ID NO: 800, SEQ IDNO: 801, SEQ ID NO: 805, SEQ ID NO: 817, SEQ ID NO: 821, SEQ ID NO: 822,SEQ ID NO: 824, SEQ ID NO: 882, SEQ ID NO: 958, SEQ ID NO: 967, SEQ IDNO: 970, and SEQ ID NO: 1027—SEQ ID NO: 1033. The alignment identifiedpermissive or preferred amino acids at a given location, and provided aguide for discovery of novel peptide variants that could be generatedand that could retain essential properties such as structure, function,peptide folding, biodistribution, or stability. SEQ ID NO: 505 and SEQID NO: 779 are consensus sequences based on the above multiple sequencealignment. SEQ ID NO: 505 is the same sequence as SEQ ID NO: 779 butwith an N-terminal “GS.” Furthermore, based on the ability to substituteK residues to R residues, the multiple sequence alignment identifiedpeptides of the family of sequences of SEQ ID NO: 506 and SEQ ID NO: 780as potential peptide variants that could be generated and that couldretain essential properties such as structure, function, peptidefolding, biodistribution, or stability. Additionally, the multiplesequence alignment identified SEQ ID NO: 796 as a conserved regionwithin the sequences of the alignment, which may, at least in part, beimportant for maintaining the essential properties such as structure,function, peptide folding, biodistribution, binding, accumulation,retention, or stability.

Example 109 Peptide Immunogenicity

This example illustrates the testing of the immunogenicity of a peptide.NetMHC II version 2.3 prediction software was used to identifyimmunogenic peptides based on a neural network alignment algorithm thatpredicts peptide binding to MHC Class II molecules.

The NetMHC II prediction software was utilized to determine the putativepeptide binding capability to DR, DQ, and DP MHC II alleles and thestrength of the interaction between peptide and MHC II molecules. TABLE57 shows the resulting immunogenicity score of select peptides. Thenumbers of strong versus weak peptides were tallied into each major MHCallele group (DR, DQ, and DP). Additionally, the numbers of ‘uniquestrong’ and ‘unique weak core’ peptides were also tallied. These datawere used to predict which peptides are less likely to induce animmunogenic response in patients. For example, the stronger a peptidebinds to an allele, the more likely it is to be presented in aMHC/peptide combination on an antigen presenting cell, thus triggeringan immune response, and a peptide that is predicted to bind to feweralleles is more likely to have weaker binding to given alleles andshould be less immunogenic.

TABLE 57 Immunogenicity Scores of Peptides Strong Binding Unique WeakBinding Alleles Strong Core Alleles Unique Weak SEQ ID NO: (DR + DQ +DP) Peptides (DR + DQ + DP) Core Peptides SEQ ID NO: 592 1 + 0 + 0 1 +0 + 0 7 + 1 + 0 7 + 2 + 0 SEQ ID NO: 744 0 + 0 + 0 0 + 0 + 0 4 + 1 + 36 + 1 + 1 SEQ ID NO: 745 0 + 0 + 0 0 + 0 + 0 4 + 1 + 3 6 + 2 + 1 SEQ IDNO: 746 1 + 0 + 0 2 + 0 + 0 5 + 1 + 3 7 + 1 + 1 SEQ ID NO: 747 1 + 0 + 02 + 0 + 0 6 + 1 + 3 5 + 1 + 1 SEQ ID NO: 748 0 + 0 + 0 0 + 0 + 0 4 + 1 +3 6 + 2 + 1 SEQ ID NO: 749 0 + 0 + 0 0 + 0 + 0 5 + 1 + 3 7 + 2 + 1 SEQID NO: 755 0 + 0 + 1 0 + 0 + 1 7 + 4 + 1 8 + 6 + 2 SEQ ID NO: 758 0 +0 + 0 0 + 0 + 0 5 + 4 + 1 5 + 4 + 2 SEQ ID NO: 882 1 + 0 + 0 1 + 0 + 07 + 1 + 0 7 + 1 + 0 SEQ ID NO: 1034 0 + 0 + 0 0 + 0 + 0 4 + 0 + 3 6 +0 + 1 SEQ ID NO: 1035 0 + 0 + 0 0 + 0 + 0 4 + 1 + 3 6 + 1 + 1 SEQ ID NO:1036 1 + 0 + 0 2 + 0 + 0 5 + 0 + 3 7 + 0 + 1 SEQ ID NO: 1037 1 + 0 + 02 + 0 + 0 6 + 0 + 3 5 + 0 + 1 SEQ ID NO: 1038 0 + 0 + 0 0 + 0 + 0 4 +1 + 3 6 + 1 + 1 SEQ ID NO: 1039 0 + 0 + 0 0 + 0 + 0 5 + 1 + 3 7 + 1 + 1SEQ ID NO: 1045 0 + 0 + 1 0 + 0 + 1 7 + 4 + 1 8 + 6 + 2 SEQ ID NO: 10480 + 0 + 0 0 + 0 + 0 5 + 4 + 1 5 + 5 + 2

Example 110 Peptide Variants

This example illustrates the design of variant peptide sequences withincreased stability, decreased regions of immunogenicity, and thesubstitution of a tyrosine for spectrophotometric reporting as comparedto a parent peptide sequence. Potential mutations to the parent peptidesequence, SEQ ID NO: 592, that may result in a peptide with increasedstability, decreased immunogenicity, or increased absorbance at 270-280nm (such as the substitution to a tyrosine or tryptophan residue forspectrophotometric reporting) were identified based on information frommultiple sequence alignment from EXAMPLE 108 and immunogenicity testingfrom EXAMPLE 109.

In SEQ ID NO: 592, residue N7 is at risk for deamidation. Based on themultiple sequence alignment of SEQ ID NO: 800, SEQ ID NO: 801, SEQ IDNO: 805, SEQ ID NO: 817, SEQ ID NO: 821, SEQ ID NO: 822, SEQ ID NO: 824,SEQ ID NO: 882, SEQ ID NO: 967, SEQ ID NO: 970, and SEQ ID NO: 1027—SEQID NO: 1033, the candidate residue mutations to best reduce this riskwere N7S and N7G. N7S was determined to be more likely to result in apeptide with desirable properties such as folding and stability as shownby matches in the alignment and conservationist presence in a peptidewith high stability (SEQ ID NO: 958).

Residue D18 is at risk for cleavage. Based on the multiple sequencealignment, the candidate residue mutations to best reduce cleavage atD18 are D18E and D18Q. D18E is the preferred choice based on retainingcharge.

Residue M25 is at risk for oxidation. Based on the multiple sequencealignment, the candidate residue mutations to best reduce oxidation wereM25T and M25A. Based on the immunogenicity score of peptides with eachmutation, it was determined that M25T is the better mutation, as iteliminates a significant source of immunogenicity as compared to SEQ IDNO: 592 as well as the variant with M25A, which did not eliminate thepredicted immunogenicity of the parent peptide of SEQ ID NO: 592.

Residue N32 is at risk for deamidation, at least in part due to theneighboring residue S33. However, N32 is conserved across Kv1. 3 bindingcystine-dense peptides in the alignment of Example 108, and implicatedin receptor binding (Peigneur, S., Biochemistry, 55(32): 2927-35(2016)). For certain applications, peptides are designed to maintainthis binding interaction, and for other applications, peptides aredesigned to remove this binding interaction. To maintain functionality,one candidate residue mutation based on the multiple sequence alignmentis S33R, which would impact deamidation. However, it resulted in apredicted increased immunogenicity score. Another candidate residuemutation is S33G, but this may result in higher deamidation rates. IfN32 is mutated, the best candidate residue mutation based the multiplesequence alignment in combination with the immunogenicity score was N32Qdespite it having a slight increase in immunogenicity. Other options areN32A, N32S, or N32T_(max) Alternatively, to remove functionality,candidate mutations based on the multiple sequence alignment are N32Aand N32L, which are the preferred choices.

For the substitution to a tyrosine for spectrophotometric reporting, thebest candidate locations were T₃₈Y (which had the strongest precedencein the multiple sequence alignment and is found in several of the stablepeptides (e.g., SEQ ID NO: 958, SEQ ID NO: 1028, and SEQ ID NO: 1029)),L17Y, and H36Y. However, T₃₈Y may slightly increase immunogenicity withrespect to the DR allele. Another option for spectrophometric absorbanceis to substitute Trp for the Leu at position 17.

Based on the above analysis, the following short list of potentialmutations for SEQ ID NO: 592 were compiled: N7S; D18E; M25T;N32Q, N32A,N32S, N32T, N32L, S33G, and S33R (variants both to retain function andto remove function of binding ion channel); and L17Y, H36Y, and T₃₈Y.

TABLE 58 provides some exemplary sequences using various combinations ofthese mutations.

TABLE 58 Exemplary Sequence Variants of SEQ ID NO: 592 SEQ ID NO:Mutations SEQ ID NO: 592 Parent SEQ ID NO: 1034 N5S, D16E, M23T, S31GSEQ ID NO: 1035 N5S, D16E, M23T, N30Q SEQ ID NO: 1036 N5S, D16E, M23T,S31R SEQ ID NO: 1037 D16E, M23T SEQ ID NO: 1038 D16E, M23T, N30Q SEQ IDNO: 1039 D16E, M23T, N30Q, T36Y SEQ ID NO: 1040 L15Y, D16E, M23T, N30QSEQ ID NO: 1041 D16E, M23T, N30Q, H34Y SEQ ID NO: 1042 N5S, D16E, M23T,N30Q, T36Y SEQ ID NO: 1043 N5S, L15Y, D16E, M23T, N30Q SEQ ID NO: 1044N5S, D16E, M23T, N30Q, H34Y SEQ ID NO: 1045 D16E, M23T, N32A, T36Y SEQID NO: 1046 D16E, M23T, N32S, T36Y SEQ ID NO: 1047 D16E, M23T, N32T,T36Y SEQ ID NO: 1048 D16E, M23T, T36Y

Example 111 Peptide-Budesonide Conjugate

This example describes conjugation of a peptide of any one of SEQ ID NO:744—SEQ ID NO: 758 or SEQ ID NO: 1034—SEQ ID NO: 1048 to budesonide.Budesonide is readily conjugated to any peptide disclosed herein viastandard chemistries such as those described in, but not limited to,Bioconjugate Techniques by Greg Hermanson (Elsevier Inc., 3^(rd)edition, 2013) or by any of the methods described in EXAMPLES 58-61.

The peptide-budesonide conjugates are administered to a subject in needthereof and home, target, are directed to, are retained by, accumulatein, migrate to, and/or bind to cartilage and/or kidneys. The subject isa human or animal and has inflammation in the cartilage or kidneytissues. Upon administration and homing of peptide-budesonideconjugates, the inflammation in the cartilage and/or kidney tissues isalleviated.

Example 112 Peptide-Dexamethasone Conjugate

This example describes conjugation of a peptide of any one of SEQ ID NO:744—SEQ ID NO: 758 or SEQ ID NO: 1034—SEQ ID NO: 1048 to dexamethasone.Dexamethasone is readily conjugated to any peptide disclosed herein viastandard chemistries such as those described in, but not limited to,Bioconjugate Techniques by Greg Hermanson (Elsevier Inc., 3^(rd)edition, 2013) or by any of the methods described in EXAMPLES 58-61.

The peptide-dexamethasone conjugates are administered to a subject inneed thereof and home, target, are directed to, are retained by,accumulate in, migrate to, and/or bind to cartilage and/or kidneys. Thesubject is a human or animal and has inflammation in the cartilage orkidney tissues. Upon administration and homing of peptide-dexamethasoneconjugates, the inflammation in the cartilage and/or kidney tissues isalleviated.

Example 113 Peptide-Triamcinalone Acetonide Conjugate

This example describes conjugation of a peptide of any one of SEQ ID NO:744—SEQ ID NO: 758 or SEQ ID NO: 1034—SEQ ID NO: 1048 to triamicinaloneacetonide. Triamicinalone acetonide is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc.^(, 3rd) edition, 2013 or by any of the methods described inEXAMPLES 58-61.

The peptide-triamicinalone acetonide conjugates are administered to asubject in need thereof and home, target, are directed to, are retainedby, accumulate in, migrate to, and/or bind to cartilage and/or kidneys.The subject is a human or animal and has inflammation in the cartilageor kidney tissues. Upon administration and homing ofpeptide-triamicinalone acetonide conjugates, the inflammation in thecartilage and/or kidney tissues is alleviated.

Example 114 Peptide-Desciclesonide Acetonide Conjugate

This example describes conjugation of a peptide of any one of SEQ ID NO:744—SEQ ID NO: 758 or SEQ ID NO: 1034—SEQ ID NO: 1048 to desciclesonideacetonide. Desciclesonide acetonide is readily conjugated to any peptidedisclosed herein via standard chemistries such as those described in,but not limited to, Bioconjugate Techniques by Greg Hermanson (ElsevierInc., 3^(rd) edition, 2013) or by any of the methods described inEXAMPLES 58-61.

The peptide-desciclesonide acetonide conjugates are administered to asubject in need thereof and home, target, are directed to, are retainedby, accumulate in, migrate to, and/or bind to cartilage and/or kidneys.The subject is a human or animal and has inflammation in the cartilageor kidney tissues. Upon administration and homing ofpeptide-desciclesonide acetonide conjugates, the inflammation in thecartilage and/or kidney tissues is alleviated.

Example 115 Method of Peptide Synthesis

This example describes the synthesis of SEQ ID NO: 590, SEQ ID NO: 592,and SEQ ID NO: 671.

A peptide of SEQ ID NO: 592 was made using Solid Phase Peptide Synthesis(SPPS). After release of the peptide from the solid phase, the peptidewas purified prior to folding by oxidation in solution. The foldedpeptide was further purified by reversed-phase chromatography andlyophilized as a TFA salt. The final SEQ ID NO: 592 peptide product hada purity of 96.1% and a mass of 4,301.7 Da, which confirmed its identityas a peptide of SEQ ID NO: 592.

A peptide of SEQ ID NO: 590 was made using Solid Phase Peptide Synthesis(SPPS). After release of the peptide from the solid phase, the peptidewas folded by oxidation in solution. The folded peptide was purified byreversed-phase chromatography and lyophilized as a TFA salt. The finalSEQ ID NO: 590 had a purity of 95.6% and a mass of 4,503.0 Da, whichconfirmed its identity as a peptide of SEQ ID NO: 590.

A peptide of SEQ ID NO: 671 was made using Solid Phase Peptide Synthesis(SPPS). After release of the peptide from the solid phase, the peptidewas folded by oxidation in solution. The folded peptide was purified byreversed-phase chromatography and lyophilized as a TFA salt. The finalSEQ ID NO: 671 peptide product had a purity of 95.5% and a mass of4,154.0 Da, which confirmed its identity as a peptide of SEQ ID NO: 671.

Example 117 Whole Body Autoradiography of Cartilage Homing Peptides

This example illustrates peptide homing to cartilage mice 5 minutes to48 hours after administration of a radiolabeled peptide. Signal from theradiolabeled peptides was found in all types of cartilage at each timepoint examined. Each peptide was radiolabeled by methylating lysines atthe N-terminus as described in EXAMPLE 35. As such, the peptide maycontain methyl or dimethyl lysines and a methylated or dimethlyatedamino terminus. A dose of 100 nmol radiolabeled peptide was administeredvia tail vein injection in Female Harlan athymic nude mice, weighing20-25 g. The experiment was done in duplicate (n=2 animals per group).Each radiolabeled peptide was allowed to freely circulate within theanimal for the described time period before the animals were euthanizedand sectioned.

Whole body autoradiography (WBA) sagittal sectioning was performed asfollows. At the end of the dosing period, mice were frozen in ahexane/dry ice bath and then embedded in a frozen block ofcarboxymethylcellulose. Whole animal sagittal slices were prepared thatresulted in thin frozen sections for imaging. Sections were allowed todessicate in a freezer prior to imaging. For the autoradiographyimaging, tape mounted thin sections were freeze dried and radioactivesamples were exposed to phosphoimager plates. These plates weredeveloped and the signal (densitometry) from each organ was normalizedto the signal found in the cardiac blood of each animal. A signal intissue darker than the signal expected from blood in that tissueindicates accumulation in a region, tissue, structure, or cell.

FIG. 47A-FIG. 47B illustrates autoradiography image of frozen sectionsfrom a mouse, 3 hours after administration of 100 nmol of a radiolabeledpeptide of SEQ ID NO: 592. FIG. 47A illustrates the ¹⁴C signal in afrozen section of a mouse, 3 hours after administration of 100 nmol of aradiolabeled peptide of SEQ ID NO: 592. The ¹⁴C signal identifies theradiolabeled peptide distribution in the cartilage of the mouse. FIG.47B illustrates the ¹⁴C signal in a different frozen section of a mouse,3 hours after administration of 100 nmol of a radiolabeled peptide ofSEQ ID NO: 592. The ¹⁴C signal identifies the radiolabeled peptidedistribution in the cartilage of the mouse.

TABLE 59 shows the signal of radiolabeled peptides of SEQ ID NO: 511 andSEQ ID NO: 592 in intervertebral discs (IVD) and knee joints as apercentage of the blood. Because the peptides may arrive at the jointwithin five minutes, a therapeutic effect from the peptide or aconjugated active agent may begin quickly. A therapeutic effect could belong lasting, due to continued presence of detected agents at 48 hoursand/or due to long lasting pharmacodynamics effects.

TABLE 59 Signal of Radiolabeled Peptides of SEQ ID NO: 511 and SEQ IDNO: 592 in IVD and Knee Joints as a Percentage of Blood SEQ ID NO: SEQID NO: SEQ ID NO: Hours 511 IVD 592 IVD 592 Knee 0.08 164 404 0.5 369510 1 961 1114 3 1779 3213 4059 8 3777 4990 24 833 5391 2137 48 3320 843

FIG. 48A-FIG. 48B illustrates autoradiography images of frozen sectionsfrom a mouse, 3 hours after administration of 100 nmol of a radiolabeledpeptide of SEQ ID NO: 590. FIG. 48A illustrates the ¹⁴C signal in afrozen section of a mouse, 3 hours after administration of 100 nmol of aradiolabeled peptide of SEQ ID NO: 590. The ¹⁴C signal identifies theradiolabeled peptide distribution in the cartilage of the mouse. FIG.48B illustrates the ¹⁴C signal in a frozen section of a different mouse,3 hours after administration of 100 nmol of a radiolabeled peptide ofSEQ ID NO: 590. The ¹⁴C signal identifies the radiolabeled peptidedistribution in the cartilage of the mouse.

FIG. 49A-FIG. 49B illustrates autoradiography images of frozen sectionsfrom a mouse, 3 hours after administration of 100 nmol of a radiolabeledpeptide of SEQ ID NO: 671. FIG. 49A illustrates the ¹⁴C signal in afrozen section of the mouse, 3 hours after administration of 100 nmol ofa radiolabeled peptide of SEQ ID NO: 671. The ¹⁴C signal identifies theradiolabeled peptide distribution in the cartilage of the mouse. FIG.49B illustrates the ¹⁴C signal in a frozen section of a different mouse,3 hours after administration of 100 nmol of a radiolabeled peptide ofSEQ ID NO: 671. The ¹⁴C signal identifies the radiolabeled peptidedistribution in the cartilage of the mouse.

This data illustrates peptides of SEQ ID NO: 511, SEQ ID NO: 592, SEQ IDNO: 590 and SEQ ID NO: 671 homed to and accumulated in the cartilage ofthe animals. The peptide of SEQ ID NO: 592 is a K to R variant of apeptide of SEQ ID NO: 511. These data show that K to R variants ofcartilage homing peptides retained their cartilage homing properties.

SEQ ID NO: 1051 (GSGVPINVRSRGSRDSLDPSRRAGMRFGRSINSRSHSTP) is alinearized version of SEQ ID NO: 592, where the knotted scaffold of thepeptide was removed by mutating out the cysteine residues that form thedisulfide bonds of the peptide to serine residues, but retaining therest of the sequence. TABLE 60 shows quantification of signal as apercentage of signal in blood from a linearized radiolabeled SEQ ID NO:1051 peptide in intervertebral discs (IVD).

TABLE 60 Signal of Radiolabeled Peptides of SEQ ID NO: 1051 in IVD as aPercentage of Blood 3 hr Ligated 3 hr Intact Kidneys 24 hr IntactKidneys IVD 117 177 104

The peptide of SEQ ID NO: 1051, a linearized version of the peptide ofSEQ ID NO: 592, homed to cartilage to a much lesser extent than thefolded knotted peptide (SEQ ID NO: 592). The signal of the foldedknotted peptide of SEQ ID NO: 592 was ˜20-fold greater at 3 hours and˜50-fold greater at 24 hours (TABLE 59) as compared to the linearizedpeptide of SEQ ID NO: 1051 (TABLE 60). These results indicate that inaddition to changes in primary sequence or peptide charge, homing tocartilage can also be related to changes in conformation, or tertiarystructure. Namely, in some cases, folded cystine-dense peptides can beexemplary cartilage homers in comparison to unfolded, linearizedpeptides of the same primary sequence (except for the mutated cysteineresidues).

Example 118 Fluorescence of Cartilage Homing Peptides

This example illustrates peptide homing to cartilage mice afteradministration of a peptide fluorophore conjugate. A peptide of SEQ IDNO: 592 was chemically conjugated to one molecule of Cyanine 5.5, andthen imaged using the methods of EXAMPLE 46.

FIG. 43A-FIG. 43F shows white light images and corresponding whole bodyfluorescence images of a mouse administered 10 nmol of a peptide of SEQID NO: 592 conjugated to a Cy5.5 fluorophore (SEQ ID NO: 592A) at 24hours post-administration. FIG. 43A illustrates an image of a frozensection of a mouse, 24 hours after administration of 10 nmol of apeptide of SEQ ID NO: 592 conjugated to a Cy5.5 fluorophore (SEQ ID NO:592A). FIG. 43B illustrates the fluorescence signal in the mouse,corresponding to the section shown in FIG. 43A, 24 hours afteradministration of 10 nmol of a peptide of SEQ ID NO: 592 conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A). FIG. 43C illustrates an image of adifferent frozen section of the mouse, 24 hours after administration of10 nmol of a peptide of SEQ ID NO: 592 conjugated to a Cy5.5 fluorophore(SEQ ID NO: 592A). FIG. 43D illustrates the fluorescence signal in themouse, corresponding to the section shown in FIG. 43C, 24 hours afteradministration of 10 nmol of a peptide of SEQ ID NO: 592 conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A). FIG. 43E illustrates an image of adifferent frozen section of the mouse, 24 hours after administration of10 nmol of a peptide of SEQ ID NO: 592 conjugated to a Cy5.5 fluorophore(SEQ ID NO: 592A). FIG. 43F illustrates a fluorescence signal in themouse, corresponding to the section shown in FIG. 43E, 24 hours afteradministration of 10 nmol of a peptide of SEQ ID NO: 592 conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A).

FIG. 46A-FIG. 46H shows IVIS fluorescence imaging of an isolated hindlimb from a first mouse and an isolated hind limb from a second mouseafter administration of 10 nmol SEQ ID NO: 592 peptide conjugated to aCy5.5 fluorophore (SEQ ID NO: 592A). FIG. 46A shows the right hind limbwith skin removed from a first mouse and from a second mouse 3 hoursafter peptide administration. FIG. 46B shows the right hind limb withmuscle removed from a first mouse and from a second mouse 3 hours afterpeptide administration. FIG. 46C shows the right hind limb with skinremoved from a first mouse and from a second mouse 24 hours afterpeptide administration. FIG. 46D shows the right hind limb with muscleremoved from a first mouse and from a second mouse 24 hours afterpeptide administration. FIG. 46E shows the right hind limb with skinremoved from a first mouse and from a second mouse 48 hours afterpeptide administration. FIG. 46F shows the right hind limb with muscleremoved from a first mouse and from a second mouse 48 hours afterpeptide administration. FIG. 46G shows the right hind limb with skinremoved from a first mouse and from a second mouse 72 hours afterpeptide administration. FIG. 46H shows the right hind limb with muscleremoved from a first mouse and from a second mouse 72 hours afterpeptide administration. Peptide fluorescence was observed in the kneejoints of isolated right hind limbs at all time points tested.

Example 119 Peptide Resistance Under Various Conditions

This example illustrates peptide stability under various stressconditions such as high temperature, low pH, reducing agents, andproteases. To determine resistance to high temperatures, cystine-densepeptides (CDPs) were incubated at 0.5 mM in PBS at 75° C. or 100° C. for1 h and pelleted, and the supernatant was analyzed with reversed-phasechromatography (RPC). To determine resistance to proteolytic digestion,CDPs were mixed with 50 U of porcine pepsin, in simulated gastric fluidat pH 1.0, or 50 U of porcine trypsin in PBS, incubated for 30 minutesat 37 ° C. and analyzed with RPC. Oxidized and reduced forms (preparedthrough addition 10 mM DTT) were compared. Circular Dichroismspectroscopy was used in order to measure the secondary structure ofpeptides with a Jasco J-720W spectropolarimeter in a cell with a 1.0-mmpath length, and CDPs were diluted into 20 mM phosphate buffer, pH 7.4,at a concentration of 15-25 μM. These conditions were expected todenature or degrade conventional globular proteins and many peptides. InTABLE 61, “high” resistance indicated a high amount of the peptideremained or was retained as unmodified under the given experimentalconditions and “low” resistance indicated a low amount of the peptideremained or was retained unmodified under the given experimentalconditions. Notably, the experimental conditions described in thisexample were more extreme stress conditions than to many standard invivo or physiologic conditions, in vitro conditions, conditions duringmanufacturing, and handling conditions. As such, even “low” resistancecan indicate meaningful resistance to these stress conditions that mayhave applicability for a number of uses described herein. The data fromthese studies are shown in TABLE 61. The peptides tested, SEQ ID NO:799, SEQ ID NO: 801 and SEQ ID NO: 966, showed high resistance to one ormore of the conditions tested.

TABLE 61 Resistance of SEQ ID NO: 801, SEQ ID NO: 799, and SEQ ID NO:966 to Various Conditions Resistance to Resistance to Resistance toResistance to Resistance to SEQ ID NO: Reduction 75° C. 100° C. PepsinTrypsin SEQ ID NO: High High High High High 511 SEQ ID NO: Low High LowHigh Low 509 SEQ ID NO: Low High Low High Low 676

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

REFERENCES

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1.-20. (canceled)
 21. A composition comprising a peptide conjugate formulated for intravenous administration, wherein the composition comprises: a buffer, wherein the buffer comprises histidine; a sugar alcohol, wherein the sugar alcohol comprises mannitol; an ionic strength of less than 50 mM; and the peptide conjugate, wherein the peptide conjugate comprises an indocyanine green conjugated to a peptide having at least 90% sequence identity to SEQ ID NO:
 9. 22. The composition of claim 21, wherein the peptide has at least 95% sequence identity to SEQ ID NO:
 9. 23. The composition of claim 21, wherein the peptide is SEQ ID NO:
 9. 24. The composition of claim 21, wherein the histidine is present at a concentration of not less than 10 mM and not greater than 100 mM.
 25. The composition of claim 21, wherein the histidine is present at a concentration of not less than 10 mM and not greater than 20 mM.
 26. The composition of claim 21, wherein the mannitol is present at a concentration of not less than 2% (wt/vol) and not greater than 20% (wt/vol).
 27. The composition of claim 21, wherein the mannitol is present at a concentration of not less than 2% (wt/vol) and not greater than 10% (wt/vol).
 28. The composition of claim 21, wherein the peptide conjugate is present at a concentration of not less than 1 mg/mL and not greater than 40 mg/mL.
 29. The composition of claim 21, wherein the peptide conjugate is present at a concentration of not less than 4 mg/mL and not greater than 10 mg/mL.
 30. The composition of claim 21, wherein the ionic strength is less than 10 mM.
 31. The composition of claim 21, comprising a pH of not less than 6.0 and not greater than 7.5.
 32. The composition of claim 21, comprising a pH of not less than 6.5 and not greater than 7.0.
 33. The composition of claim 21, comprising: the peptide conjugate at a concentration of not less than 4 mg/mL and not greater than 10 mg/mL; the histidine at a concentration of not less than 10 mM and not greater than 20 mM; the mannitol at a concentration of not less than 2% (wt/vol) and not greater than 10% (wt/vol); the ionic strength of less than 10 mM; and a pH of not less than 6.5 and not greater than 7.0.
 34. A lyophilized composition comprising: a peptide conjugate, wherein the peptide conjugate comprises an indocyanine green conjugated to a peptide having at least 90% sequence identity to SEQ ID NO: 9; a buffer, wherein the buffer comprises histidine; and a sugar alcohol, wherein the sugar alcohol comprises mannitol; wherein, upon reconstitution of the lyophilized composition with water, an aqueous solution comprising an ionic strength of less than 50 mM and a pH of not less than 6.0 and not greater than 7.5 is produced.
 35. The lyophilized composition of claim 34, wherein the mannitol is present at a concentration of not less than 2% (wt/vol) and not greater than 20% (wt/vol) in the aqueous solution.
 36. The lyophilized composition of claim 34, wherein the histidine is present at a concentration of not less than 10 mM and not greater than 100 mM in the aqueous solution.
 37. The lyophilized composition of claim 34, wherein the peptide conjugate is present at a concentration of not less than 1 mg/mL and not greater than 40 mg/mL in the aqueous solution.
 38. The lyophilized composition of claim 34, wherein the aqueous solution comprises a pH of not less than 6.0 and not greater than 7.5. 